DNA encoding enzyme, recombinant DNA and enzyme, transformant, and their preparation and uses

ABSTRACT

Disclosed are a DNA encoding an enzyme which releases trehalose from non-reducing saccharides having a trehalose structure as an end unit and having a degree of glucose polymerization of 3 or higher, recombinant DNA and enzyme, transformant, and their preparations and uses. These facilitate the industrial-scale production of trehalose with a relative easiness and low cost, and trehalose thus obtained can be satisfactorily used in a variety of food products, cosmetics and pharmaceuticals.

CROSS REFERENCE TO RELATED APPLICATIONS

This present application is a division of U.S. application Ser. No.08/399,646, filed Mar. 7, 1995, now U.S. Pat. No. 5,556,781.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a novel DNA encoding an enzyme whichreleases trehalose from non-reducing saccharides having a trehalosestructure as an end unit and having a degree of glucose polymerizationof 3 or higher, recombinant DNA containing the same, and a transformant,and further relates to a recombinant enzyme which releases trehalosefrom non-reducing saccharides having a trehalose structure as an endunit and having a degree of glucose polymerization of 3 or higher, aswell as to preparations and uses thereof.

2. Description of the Prior Art

Trehalose is a disaccharide which consists of 2 glucose molecules whichare linked together with their reducing groups, and, naturally, it ispresent in bacteria, fungi, algae, insects, etc., in an extremely smallquantity. Having no reducing residue within the molecule, trehalose doesnot cause an unsatisfactory browning reaction even when heated in thepresence of amino acids or the like, and because of this it can sweetenfood products without fear of causing unsatisfactory coloration anddeterioration. Trehalose, however, is far from being readily prepared ina desired amount by conventional methods, and, actually, it has notscarcely been used for sweetening food products.

Conventional methods are roughly, classified into 2 groups, i.e. the oneusing cells of microorganisms and the other employing a multi-enzymaticsystem wherein enzymes are allowed to act on saccharides. The former, asdisclosed in Japanese Patent Laid-Open No.154,485/75, is a method whichcomprises allowing to grow microorganisms such as bacteria and yeasts ina nutrient culture medium, and collecting trehalose from theproliferated cells in the resultant culture. The latter, as disclosed inJapanese Patent Laid-Open No.216,695/83, is a method which comprisesproviding maltose as a substrate, allowing a multi-enzymatic systemusing maltose- and trehalose-phosphorylases to act on maltose, andisolating the formed trehalose from the reaction system. Although theformer facilitates the growth of microorganisms with a relativeeasiness, it requires a sequentially-complicated step for collectingtrehalose from the microorganisms which contain at most 15 w/w%trehalose, on a dry solid basis (d.s.b.). While the latter enables theseparation of trehalose itself with a relative easiness, but it istheoretically difficult to increase the trehalose yield by allowingenzymes to act on substrates at a considerably-high concentrationbecause the enzymatic reaction per se is an equilibrium reaction of 2different types of enzymes and the equilibrium point constantly inclinesto the side of forming glucose phosphate.

In view of the foregoing, the present inventors energetically screenedenzymes which form saccharides having a trehalose structure fromamylaceous saccharides, and found that microorganisms such as those ofthe species Rhizobium sp. M-11 and Arthrobacter sp. Q36 produce anabsolutely novel enzyme which forms non-reducing saccharides having atrehalose structure as an end unit from reducing amylaceous saccharideshaving a degree of glucose polymerization of 3 or higher. Before orafter this finding, it was revealed that such non-reducing saccharidesare almost quantitatively hydrolyzed into trehalose and glucose and/ormaltooligosaccharides by other enzymes produced from the samemicroorganisms of the species Rhizobium sp. M-11 and Arthrobacter sp.Q36. Since the combination use of such enzymes enables to form a-desiredamount of trehalose with a relative easiness, the aforementioned objectsrelating to trehalose would be completely overcome. Insufficientproducibility of such enzymes by the microorganisms results in adrawback that a relatively-large scale culture of the microorganisms isinevitable to industrially produce trehalose and/or non-reducingsaccharides having a trehalose structure as an end unit.

Recombinant DNA technology has made a remarkable progress in recentyears. At present, even an enzyme, whose total amino acid sequence hasnot yet been revealed, can be readily prepared in a desired amount, if agene encoding the enzyme was once isolated and the base sequence wasdecoded, by preparing a recombinant DNA containing a DNA which encodesthe enzyme, introducing the recombinant DNA into microorganisms or cellsof plants or animals, and culturing the resultant transformants. Underthese circumstances, urgently required are the finding of genes whichencode these enzymes and the elucidation of their base sequences.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a DNA which encodesan enzyme that releases trehalose from non-reducing saccharides having atrehalose structure as an end unit.

It is a further object of the present invention to provide a replicablerecombinant DNA containing the aforesaid DNA.

It is yet another object of the present invention to provide atransformant which is prepared by introducing the recombinant DNA intoan appropriate host.

It is a further object of the present invention to prepare the aforesaidenzyme by the application of the recombinant DNA technology.

It is a further object of the present invention to provide a preparationof the enzyme.

It is a further object of the present invention to provide a method forconverting non-reducing saccharides having a trehalose structure as anend unit and having a degree of glucose polymerization of 3 or higher.

The first object of the present invention is attained by a DNA whichencodes an enzyme that releases trehalose from non-reducing saccharideshaving a trehalose structure as an end unit and having a degree ofglucose polymerization of 3 or higher.

The second object of the present invention is attained by a replicablerecombinant DNA which contains the aforesaid DNA and a self-replicablevector.

The third object of the present invention is attained by a transformantprepared by introducing the aforesaid self-replicable vector into anappropriate host.

The fourth object of the present invention is attained by a recombinantenzyme which releases trehalose from non-reducing saccharides having atrehalose structure as an end unit and having a degree of glucosepolymerization of 3 or higher.

The fifth object of the present invention is attained by a process toproduce the recombinant enzyme comprising culturing a transformantcapable of forming the enzyme in a nutrient culture medium, andrecovering the formed enzyme from the resultant culture.

The sixth object of the present invention is attained by a method forconverting non-reducing saccharides containing a step of allowing therecombinant enzyme to act on non-reducing saccharides, having atrehalose structure as an end unit and having a degree of glucosepolymerization of 3 or higher, to release trehalose from thesaccharides.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

FIG. 1 shows the optimum temperature of an enzyme derived from Rhizobiumsp. M-11.

FIG. 2 shows the optimum temperature of an enzyme derived fromArthrobacter sp. Q36.

FIG. 3 shows the optimum pH of an enzyme derived from Rhizobium sp.M-11.

FIG. 4 shows the optimum pH of an enzyme derived from Arthrobacter sp.Q36.

FIG. 5 shows the thermal stability of an enzyme derived from Rhizobiumsp. M-11.

FIG. 6 shows the thermal stability of an enzyme derived fromArthrobacter sp. Q36.

FIG. 7 shows the pH stability of an enzyme derived from Rhizobium sp.M-11.

FIG. 8 shows the pH stability of an enzyme derived from Arthrobacter sp.Q36.

FIG. 9 shows the restriction map of the recombinant DNA pBMU27 accordingto the present invention. In the figure, the bold-lined part is a DNAencoding an enzyme derived from Rhizobium sp. M-11.

FIG. 10 shows the restriction map of the recombinant DNA pBRT32according to the present invention. In the figure, the bold-lined partis a DNA encoding an enzyme derived from Arthrobacter sp. Q36.

DETAILED DESCRIPTION OF THE INVENTION

The DNA according to the present invention exerts the production of theenzyme encoded by the DNA in a manner that the DNA is inserted into anappropriate self-replicable vector to form a replicable recombinant DNA,followed by introducing the recombinant DNA into a host, incapable ofproducing the enzyme per se but readily replicable, to form atransformant.

Although the recombinant DNA per se does not produce the enzyme, theproduction of the enzyme encoded by the DNA is attained by introducingthe recombinant DNA into a host, incapable of producing the enzyme butreplicable with a relative easiness, to form a transformant, andculturing the transformant to produce the enzyme.

The transformant according to the present invention produces the enzymewhen cultured.

The recombinant enzyme according to the present invention releasestrehalose when acts on non-reducing saccharides having a trehalosestructure as an end unit and having a degree of glucose polymerizationof 3 or higher.

The recombinant enzyme is readily obtained in a desired amount byculturing the transformant according to the invention.

Non-reducing saccharides having a trehalose structure as an end unit andhaving a degree of glucose polymerization of 3 or higher are convertedinto trehalose and glucose and/or maltooligosaccharides.

The present invention is based on the finding of a novel enzyme whichreleases trehalose from non-reducing saccharides having a trehalosestructure as an end unit and having a degree of glucose polymerizationof 3 or higher. Such an enzyme can be obtained from cultures ofmicroorganisms of the species Rhizobium sp. M-11 and Arthrobacter sp.Q36, and the present inventors isolated the enzyme by the combinationuse of conventional purification methods using column chromatographymainly, examined the properties and features, and revealed the reality,i.e. it is a polypeptide having the following physicochemicalproperties:

(1) Action Releasing trehalose from non-reducing saccharides having atrehalose structure as an end unit and having a degree of glucosepolymerization of 3 or higher;

(2) Molecular weight About 57,000-68,000 daltons on sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE);

(3) Isoelectric point About 3.3-4.6 on isoelectrophoresis;

(4) Optimum temperature Exhibiting an optimum temperature of around35°-45° C. when incubated at pH 7.0 for 30 min;

(5) Optimum pH Exhibiting an optimum pH of around 6.0-7.5 when incubatedat 40° C. for 30 min;

(6) Thermal stability Stable up to a temperature of around 30°-45° C.when incubated at pH 7.0 for 60 min; and

(7) pH Stability Stable up to a pH of around 5.5-10.0 when incubated at25° C. for 16 hours.

Experiments, which were conducted to reveal the physicochemicalproperties of the enzymes produced by microorganisms of the speciesRhizobium sp. M-11 and Arthrobacter sp. Q36 (the enzymes from Rhizobiumsp. M-11 and Arthrobacter sp. Q36 are respectively designated as "enzymeM-11" and "enzyme Q36" hereinafter), are explained in the below:

EXPERIMENT 1 Purification of enzyme EXPERIMENT 1-1 Purification ofenzyme M-11

In 500-ml Erlenmeyer flasks were placed 100 ml aliquots of a liquidculture medium (pH 7.0) containing 2.0 w/v % "PINE-DEX #4", a starchhydrolysate commercialized by Matsutani Chemical Ind., Co., Ltd., Tokyo,Japan, 0.5 w/v % peptone, 0.1 w/v % yeast extract, 0.1 w/v % disodiumhydrogen phosphate, and 0.1 w/v % potassium dihydrogen phosphate, andthe flasks were autoclaved at 120° C. for 20 min to effectsterilization. After cooling the flasks a seed culture of Rhizobium sp.M-11 was inoculated into each liquid culture medium in each flask,followed by the incubation at 27° C. for 24 hours under rotary-shakingconditions. Twenty L of a fresh preparation of the same liquid culturemedium was put in a 30-L jar fermentor and sterilized, followed byinoculating one v/v % of the culture obtained in the above into thesterilized liquid culture medium in the jar fermentor, and incubating itat a pH of 6-8 and 30° C. for 24 hours under aeration-agitationconditions.

Thereafter, about 18 L of the resultant culture was subjected to anultra-high pressure cell disrupting apparatus to disrupt cells. Theresultant suspension was centrifuged to obtain a supernatant, and toabout 16 L of which was added ammonium sulfate to give a 20 w/v %saturation, followed by the standing at 4° C. for one hour and thecentrifugation to remove sediment. To the resultant supernatant wasadded ammonium sulfate to give a 60 w/v % saturation, and the solutionwas allowed to stand at 4° C. for 24 hours and centrifuged to collectsediment which was then dissolved in a minimum amount of 10 mM phosphatebuffer (pH 7.0). The solution thus obtained was dialyzed against 10 mMphosphate buffer (pH 7.0) for 24 hours, and centrifuged to removeinsoluble substances. The resultant supernatant was fed to a columnpacked with "DEAE-TOYOPEARL®", a product for ion-exchange chromatographycommercialized by Tosoh Corporation, Tokyo, Japan, which had beenpreviously equilibrated with 10 mM phosphate buffer (pH 7.0), followedby feeding to the column a linear gradient buffer of sodium chlorideranging from 0 M to 0.5 M in 10 mM phosphate buffer (pH 7.0). Fractionscontaining the objective enzyme were collected from the eluate, pooled,dialyzed for 10 hours against 50 mM phosphate buffer (pH 7.0) containing2 M ammonium sulfate, and centrifuged to remove insoluble substances.Thereafter, the resultant supernatant was fed to a column, which hadbeen packed with "BUTYL TOYOPEARL®", a gel for hydrophobic columnchromatography commercialized by Tosoh Corporation, Tokyo, Japan, andequilibrated with 50 mM phosphate buffer (pH 7.0) containing 2 Mammonium sulfate, followed by feeding to the column a linear gradientbuffer of ammonium sulfate ranging from 2 M to 0 M in 50 mM phosphatebuffer (pH 7.0). Fractions containing the objective enzyme werecollected from the eluate, pooled, fed to a column packed with"TOYOPEARL® HW-55", a product for gel filtration column chromatographycommercialized by Tosoh Corporation, Tokyo, Japan, which had beenpreviously equilibrated with 50 mM phosphate buffer (pH 7.0), followedby feeding to the column 50 mM phosphate buffer (pH 7.0) and collectingfractions containing the objective enzyme. The enzyme thus obtained hada specific activity of about 240 units/mg protein, and the yield wasabout 650 units per L of the culture.

Throughout the specification the enzyme activity is expressed by thevalue measured on the following assay: Place 4 ml of 50 mM phosphatebuffer (pH 7.0) containing 1.25 w/v % maltotriosyltrehalose in a testtube, add one ml of an enzyme solution to the tube, and incubate theresultant solution at 40° C. for 30 min to effect enzymatic reaction.Thereafter, one ml of the reaction mixture is mixed with 2 ml of copperreagent to suspend the enzymatic reaction, followed by assaying thereducing activity by the Somogyi-Nelson's method. As a control, anenzyme, which has been previously inactivated by heating at 100° C. for10 min, is similarly treated as above. One unit activity of the enzymeis defined as the amount of enzyme which increases the reducing powercorresponding to one μmol glucose per min under the above conditions.

EXPERIMENT 1-2 Purification of enzyme 036

Similarly as in Experiment 1-1, a seed culture of Arthrobacter sp. Q36was cultured, and the resultant culture was treated to obtain a purifiedenzyme Q36 having a specific activity of about 450 units/mg protein in ayield of about 650 units per L of the culture.

EXPERIMENT 2 Physicochemical property of enzyme EXPERIMENT 2-1 Action

According to the method disclosed in Japanese Patent ApplicationNo.349,216/93, a non-reducing saccharide containing 98 W/W % or higher,d.s.b., α-glucosyltrehalose, α-maltosyltrehalose,α-maltotriosyltrehalose, α-maltotetraosyltrehalose orα-maltopentaosyltrehalose. Either of the non-reducing saccharides as asubstrate was dissolved in 50 mM phosphate buffer (pH 7.0) into a 20 w/v% solution which was then mixed with 2 units/g substrate of the purifiedenzyme M-11 or Q36 in Experiment 1 and subjected to an enzymaticreaction at 40° C. for 48 hours. The reaction mixture was desalted inusual manner, fed to "WB-T-330", a column for high-performance liquidchromatography (HPLC) commercialized by Wako Pure Chemical Industries,Ltd., Tokyo, Japan, followed by feeding to the column distilled water ata flow rate of 0.5 ml/min at ambient temperature to isolate saccharidescontained in the reaction mixture while monitoring the saccharideconcentration of the eluate with "MODEL RI-8012", a differentialrefractometer commercialized by Tosoh Corporation, Tokyo, Japan. As acontrol, an aqueous solution which contains either maltotriose,maltotetraose, maltopentaose, maltohexaose or maltoheptaose wassimilarly treated as above, and the resultant mixture was analyzed. Thesaccharide compositions of the reaction mixtures were tabulated inTables 1 and 2.

                  TABLE 1                                                         ______________________________________                                                                      Saccharide                                                    Saccharide in   composition                                     Substrate     reaction mixture                                                                              (%)                                             ______________________________________                                                      Trehalose       17.5                                            α-Glucosyltrehalose                                                                   Glucose         6.5                                                           α-Glucosyltrehalose                                                                     76.0                                                          Trehalose       44.3                                            α-Maltosyltrehalose                                                                   Maltose         44.4                                                          α-Maltosyltrehalose                                                                     11.3                                                          Trehalose       39.5                                            α-Maltotriosyltrehalose                                                               Maltotriose     60.0                                                          α-Maltotriosyltrehalose                                                                 0.5                                                           Trehalose       34.2                                            α-Maltotetraosyltrehalose                                                             Maltotetraose   65.5                                                          α-Maltotetraosyltrehalose                                                               0.3                                                           Trehalose       29.1                                            α-Maltopentaosyltrehalose                                                             Maltopentaose   70.6                                                          α-Maltopentaosyltrehalose                                                               0.3                                             Maltotriose   Maltotriose     100.0                                           Maltotetraose Maltotetraose   100.0                                           Maltopentaose Maltopentaose   100.0                                           Maltohexaose  Maltohexaose    100.0                                           Maltoheptaose Maltoheptaose   100.0                                           ______________________________________                                    

                  TABLE 2                                                         ______________________________________                                                                      Saccharide                                                    Saccharide in   Composition                                     Substrate     reaction mixture                                                                              (%)                                             ______________________________________                                                      Trehalose       19.3                                            α-Glucosyltrehalose                                                                   Glucose         10.2                                                          α-Glucosyltrehalose                                                                     70.5                                            α-Maltosyltrehalose                                                                   Trehalose       44.5                                                          Maltose         44.4                                                          α-Maltosyltrehalose                                                                     11.1                                                          Trehalose       38.8                                            α-Maltotriosyltrehalose                                                               Maltotriose     60.7                                                          α-Maltotriosyltrehalose                                                                 0.5                                                           Trehalose       34.1                                            α-Maltotetraosyltrehalose                                                             Maltotetraose   65.7                                                          α-Maltotetraosyltrehalose                                                               0.2                                                           Trehalose       29.3                                            α-Maltopentaosyltrehalose                                                             Maltopentaose   70.4                                                          α-Maltopentaosyltrehalose                                                               0.3                                             Maltotriose   Maltotriose     100.0                                           Maltotetraose Maltotetraose   100.0                                           Maltopentaose Maltopentaose   100.0                                           Maltohexaose  Maltohexaose    100.0                                           Maltoheptaose Maltoheptaose   100.0                                           ______________________________________                                    

As shown in Tables 1 and 2, enzymes M-11 and Q36 almost quantitativelyreleased trehalose, glucose and maltooligosaccharides from non-reducingsaccharides having a trehalose structure as an end unit and having adegree of glucose polymerization of 3 or higher. These enzymes did notact on maltooligosaccharides, as a substrate, having a degree of glucosepolymerization of 3 or higher. These facts indicate that these enzymesselectively act on non-reducing saccharides having a trehalose structureas an end unit and having a degree of polymerization degree of 3 orhigher, and specifically hydrolyze the glycosidic bond betweentrehalose- and glycosyl-residues. Such an enzyme has never been reportedand is estimated to have a novel enzymatic reaction mechanism.

EXPERIMENT 2-2 Molecular weight

In accordance with the method reported by U. K. Laemmli in Nature,Vol.227, pp.680-685 (1970), the purified enzymes M-11 and Q36 inExperiment 1 were respectively electrophoresed on sodium dodecyl sulfatepolyacrylamide gel electrophoresis to show a single protein band at aposition corresponding to about 57,000-68,000 daltons. The markerproteins used in this experiment were myosin (MW=200,000 daltons),β-galactosidase (MW=116,250 daltons), phosphorylase B (MW=97,400daltons), serum albumin (MW=66,200 daltons) and ovalbumin (MW=45,000daltons).

EXPERIMENT 2-3 Isoelectric point

The purified enzymes M-11 and Q36 obtained in Experiment 1 gave anisoelectric point of about 3.3-4.6 on isoelectrophoresis.

EXPERIMENT 2-4 Optimum temperature

The optimum temperature of the purified enzymes M-11 and Q36 obtained inExperiment 1 was about 35°-45° C. as shown in FIGS. 1 and 2 whenincubated in usual manner in 50 mM phosphate buffer (pH 7.0) for 30 min.

EXPERIMENT 2-5 Optimum pH

The optimum pH of the purified enzymes M-11 and Q36 obtained inExperiment 1 was about 6.0-7.5 as shown in FIGS. 3 and 4 whenexperimented in usual manner by incubating them at 40° C. for 30 min in50 mM acetate buffer, phosphate buffer or sodium carbonate-sodiumhydrogen carbonate buffer having different pHs.

EXPERIMENT 2-6 Thermal stability

The purified enzymes M-11 and Q36 obtained in Experiment 1 were stableup to a temperature of about 30°-45° C. as shown in FIGS. 5 and 6 whenexperimented in usual manner by incubating them in 50 mM phosphatebuffer (pH 7.0) for 60 min.

EXPERIMENT 2-7 pH Stability

The purified enzymes M-11 and Q36 obtained in Experiment 1 were stableup to a pH of about 5.5-10.0 as shown in FIGS. 7 and 8 when experimentedin usual manner by incubating them at 25° C. for 16 hours in 50 mMacetate buffer, phosphate buffer or sodium carbonate-sodium hydrogencarbonate buffer having different pHs.

EXPERIMENT 2-8 Amino acid sequence containing the N-terminal

The amino acid sequence containing the N-terminal of the purified enzymeM-11 obtained in Experiment 1 was analyzed on "MODEL 470A", a gas-phaseprotein sequencer commercialized by Applied Biosystems, Inc., FosterCity, USA, to reveal that it has the amino acid sequence as shown in SEQID NO:5.

The amino acid sequence containing the N-terminal of the purified enzymeQ36 was analyzed similarly as above to reveal that it has the amino acidsequence as shown in SEQ ID NO:6.

EXPERIMENT 2-9 Partial amino acid sequence

An adequate amount of the purified enzyme M-11 obtained in Experiment1-1 was weighed, dialyzed against 10 mM Tris-HCl buffer (pH 9.0) at 4°C. for 18 hours, and admixed with 10 mM Tris-HCl buffer (pH 9.0) to givea concentration of about one mg/ml of the enzyme. About one ml of theresultant solution was placed in a container, admixed with 10 μg lysylendopeptidase, and incubated at 30° C. for 22 hours to partiallyhydrolyze the enzyme. The resultant hydrolysate was applied to"CAPCELL-PAK C18", a column for reverse-phase high-performance liquidchromatography commercialized by Shiseido Co., Ltd., Tokyo, Japan, whichhad been previously equilibrated with 0.1 v/v % trifluoroacetatecontaining 16 v/v % aqueous acetonitrile, followed by feeding to thecolumn 0.1 v/v % trifluoroacetate at a flow rate of 0.9 ml/min whileincreasing the concentration of acetonitrile from 16 v/v % to 64 v/v %to separately collect fractions containing a peptide fragment elutedabout 43 min or about 57 min after the initiation of feeding (thepeptide fragments were respectively named "peptide fragment A" and"peptide fragment B"). Fractions containing the peptide fragment A or Bwere separately pooled, dried in vacuo, and dissolved in 0.1 v/v %trifluoroacetate containing 50 v/v % aqueous acetonitrile. Similarly asin Experiment 2-8, the peptide fragments A and B were analyzed to revealthat they have the amino acid sequences as shown in SEQ ID NOs:7 and 8,respectively.

Similarly as in enzyme M-11, enzyme Q36 obtained in Experiment 1-2 waspartially hydrolyzed, and the resultant was fed to "μBONDAPAK C18", acolumn for reverse-phase high-performance liquid chromatographycommercialized by Japan Millipore Ltd., Tokyo, Japan, which had beenpreviously equilibrated with 0.1 v/v % trifluoroacetate containing 24v/v % aqueous acetonitrile, followed by feeding to the column 0.1 v/v %trifluoroacetate containing 24 v/v % aqueous acetonitrile whileincreasing the concentration of aqueous acetonitrile from 24 v/v % to 44v/v % at a flow rate of 0.9 ml/ml. Fractions containing a peptidefragment eluted about 4 min or about 24 min after the initiation offeeding (the fractions were respectively called "peptide fragment C" and"peptide fragment D" hereinafter) were respectively collected, pooled,dried in vacuo, and dissolved in 0.1 v/v % trifluoroacetate containing50 v/v % aqueous acetonitrile. Analyses of the peptide fragments C and Dconducted similarly as above have revealed that they have amino acidsequences as shown in SEQ ID NOs:9 10 respectively.

No enzyme having these physicochemical properties has been known, andthis concluded that it is a novel substance. Referring to Rhizobium sp.M-11, it is a microorganism which was isolated from a soil ofOkayama-city, Okayama, Japan, deposited on Dec. 24, 1992, in NationalInstitute of Bioscience and Human-Technology Agency of IndustrialScience and Technology, Tsukuba, Ibaraki, Japan, and accepted under theaccession number of FERM BP-4130, and it has been maintained by theinstitute. Arthrobacter sp. Q36 is a microorganism which was isolatedfrom a soil of Soja-city, Okayama, Japan, deposited on Jun. 3, 1993, inthe same institute, and accepted under the accession number of FERMBP-4316, and it has been maintained by the institute.

Japanese Patent Application No.340,343/93, applied by the sameapplicant, discloses the properties and features of the non-reducingsaccharide-forming enzyme as well as the detailed bacteriologicalproperties of these microorganisms.

The present inventors energetically screened the chromosomal DNA ofRhizobium sp. M-11 by using an oligonucleotide as a probe which had beenchemically synthesized based on the partial amino acid sequence ofenzyme M-11 as revealed in Experiment 2-8 or 2-9, and obtained a DNAfragment which consists of 1,767 base pairs having the base sequence asshown in the following SEQ ID NO:1 that initiates from the 5'-terminus.The decoding of the base sequence of the enzyme has revealed that it hasan amino acid sequence consisting of 589 amino acids as shown in SEQ IDNO:2.

Similarly as in enzyme M-11, the chromosomal DNA of enzyme Q36 wasscreened by using an oligonucleotide as a probe which had beenchemically synthesized based on a partial amino acid sequence of enzymeQ36, and this yielded a DNA fragment having a base sequence consistingof 1,791 base pairs as shown in SEQ ID NO:3. The base sequence wasdecoded to reveal that enzyme Q36 has an amino acid sequence consistingof 597 amino acids as shown in SEQ ID NO:4.

The sequential experimental steps used for revealing the base sequenceand amino acid sequence as shown in SEQ ID NOs:1 to 4 are summarized asbelow:

(1) The enzyme was isolated from a culture of a donor microorganism andhighly purified. The purified enzyme was partially hydrolyzed withprotease, and the resultant 2 different types of peptide fragments wereisolated and determined their amino acid sequences;

(2) Separately, a chromosomal DNA was isolated from a donormicroorganism's cell, purified and partially digested by a restrictionenzyme to obtain a DNA fragment consisting of about 2,000-6,000 basepairs. The DNA fragment was ligated by DNA ligase to a plasmid vector,which had been previously cut with a restriction enzyme, to obtain arecombinant DNA;

(3) The recombinant DNA was introduced into Escherichia coli to obtaintransformants, and from which an objective transformant containing a DNAencoding the enzyme was selected by the colony hybridization methodusing an oligonucleotide, as a probe, which had been chemicallysynthesized based on the aforesaid partial amino acid sequence; and

(4) The recombinant DNA was obtained from the selected transformant andannealed with a primer, followed by allowing a DNA polymerase to act onthe resultant to extend the primer, and determining the base sequence ofthe resultant complementary chain DNA by the dideoxy chain terminationmethod. The comparison of an amino acid sequence, estimable from thedetermined base sequence with the aforesaid amino acid sequence,confirmed that the base sequence encodes the enzyme.

The recombinant enzyme as referred to in the specification mean thewhole recombinant enzymes which are preparable by the recombinant DNAtechnology and capable of releasing trehalose from non-reducingsaccharides having a trehalose structure as an end unit and having adegree of glucose polymerization of 3 or higher. Generally, therecombinant enzyme according to the present invention has a revealedamino acid sequence, and, as an example, the amino acid sequence asshown in SEQ ID NO:2 or 4 which initiates from the N-terminal, as wellas homologous ones to it, can be mentioned. Variants having amino acidsequences homologous to the one as shown in SEQ ID NO:2 or 4 can beobtained by replacing one or more amino acids in SEQ ID NO:2 or 4 withother amino acids without substantially altering the inherent activityof the enzyme. Although even when used the same DNA and it also dependson hosts into which the DNA is introduced, as well as on ingredients andcomponents of nutrient culture media used for culturing transformants,and their cultivation temperature and pH, there may be produced modifiedenzymes which have amino acid sequences similar to that of SEQ ID NO:2or 4, as well as having the enzymatic activity inherent to the enzymeencoded by the DNA but defective one or more amino acids located near tothe N-terminal of the amino acid sequence of SEQ ID NO:2 or 4 and/orhaving one or more amino acids newly added to the N-terminal by themodification of intracellular enzymes of hosts after the DNA expression.In view of the technical background in the art, the enzyme as referredto in the present invention includes those which have the amino acidsequence corresponding to that of SEQ ID NO:2 or 4, and those whichsubstantially have the amino acid sequence as shown in SEQ ID NO: 2 or 4except that one or more amino acids in the amino acid sequence aredefected, newly added to or replaced with other amino acids, as long asthey release trehalose form non-reducing saccharides having a trehalosestructure as an end unit and having a degree of glucose polymerizationof 3 or higher.

In this field, it is known that one or more bases in DNAs can bereplaced with other bases by the degeneracy of genetic code withoutaltering the amino acid sequences encoded by the DNAs. Based on this theDNA according to the present invention includes DNAs which contain thebase sequence of SEQ ID NO:1 or 3 and other DNAs, wherein one or morebases are replaced with other bases by degeneracy of genetic code, aslong as they encode enzymes having the amino acid sequence as shown inSEQ ID NO: 2 or 4 and homologous variants thereof.

According to the today's recombinant DNA technology, the determinationof base sequences from the 5'-termini of DNAs define their complementarybase sequences. Therefore, the DNA according to the present inventionalso includes complementary base sequences corresponding to any one ofthe aforesaid base sequences. Needless to say, one or more bases in thebase sequence, which encodes the enzyme or their variants, can bereadily replaced with other bases to allow the DNA to actually expressthe enzyme production in hosts.

The DNA according to the present invention is as described above, andany DNA derived from natural resources and those artificiallysynthesized can be used in the present invention as long as they havethe aforementioned base sequences. The natural resources of the DNAaccording to the present invention are, for example, microorganisms ofthe genera Rhizobium, Arthrobacter, Brevibacterium and Micrococcus, i.e.Rhizobium sp. M-11 (FERM BP-4130), Arthrobacter sp. Q36 (FERM BP-4316),Brevibacterium helovolum (ATCC 11822) and Micrococcus roseus (ATCC 186)from which genes containing the present DNA can be obtained. Thesemicroorganisms can be inoculated in nutrient culture media and culturedfor about 1-3 days under aerobic conditions, and the resultant cellswere collected from the cultures and subjected to ultrasonication ortreated with a cell-wall lysis enzyme such as lysozyme or β-glucanase toextract genes containing the present DNA. In this case, a proteolyticenzyme such as protease can be used along with the cell-wall lysisenzyme, and, in the case of treating the cells with ultrasonication,they may be treated in the presence of a surfactant such as sodiumdodecyl sulfate (SDS) or treated with freezing- and thawing-methods. Theobjective DNA is obtainable by treating the resultant with phenolextraction, alcohol sedimentation, centrifugation, protease treatmentand/or ribonuclease treatment used in general in the art.

To artificially synthesize the DNA according to the present invention,it can be chemically synthesized by using the base sequence as shown inSEQ ID NO:1 or 3, or can be obtained in plasmid form by inserting a DNA,which encodes the amino acid sequence as shown in SEQ ID NO:2 or 4, intoan appropriate self- replicable vector to obtain a recombinant DNA,introducing the recombinant DNA into an appropriate host to obtain atransformant, culturing the transformant, separating the proliferatedcells from the resultant culture, and collecting plasmids containing theDNA from the cells.

The present invention further relates to replicable recombinant DNAswhich express the production of the enzyme according to the inventionwhen introduced into microorganisms as well as plant- and animal-cellswhich do not produce the enzyme inherently but are readilyproliferative. Such a recombinant DNA, which generally contains theaforesaid DNA and a self-replicable vector, can be prepared byconventional method with a relative easiness when the material DNA is inhand. Examples of such a vector are plasmid vectors such as pBR322,pUC18, Bluescript II SK(+), pUB110, pTZ4, pC194, pHV14, TRp7, TEp7,pBS7, etc.; and phage vectors such as λgt·λC, λgt·λB, ρ11, φ1, φ105,etc. Among these plasmid- and phage-vectors, pBR322, pUC18, BluescriptII SK(+), λgt·λC and λgt·λB are satisfactorily used in case that thepresent DNA should be expressed in Escherichia coli, while pUB110, pTZ4,pC194, ρ11, φ1 and φ105 are satisfactorily used to express the DNA inmicroorganisms of the genus Bacillus. The plasmid vectors pHV14, TRp7,TEp7 and pBS7 are suitably used when the recombinant DNA is allowed togrow in 2 or more hosts.

The methods used to insert the present DNA into such vectors in thepresent invention may be conventional ones generally used in this field.A gene containing the present DNA and a self-replicable vector are firstdigested by a restriction enzyme and/or ultrasonic disintegrator, thenthe resultant DNA fragments and vector fragments are ligated. To digestDNAs and vectors, restriction enzymes which specifically act onnucleotides, particularly, type II restriction enzymes, moreparticularly, Sau 3AI, Eco RI, Hind III, Bam HI, Sal I, Xba I, Sac I,Pst I, etc., facilitate the ligation of the DNA fragments and vectorfragments. The ligation of the DNA fragments and vector fragments iseffected by annealing them first if necessary, then subjected to theaction of a DNA ligase in vivo or in vitro. The recombinant DNA thusobtained is replicable without substantial limitation by introducing itinto appropriate hosts, and culturing the resultant transformants.

The recombinant DNA according to the present invention can be introducedinto appropriate host microorganisms including Escherichia coli andthose of the genus Bacillus as well as actinomyces and yeasts. In thecase of using Escherichia coli as a host, it can be cultured in thepresence of the recombinant DNA and calcium ion, while in the case ofusing the microorganisms of the genus Bacillus the competent cell methodand the colony hybridization method can be employed. Desiredtransformants can be cloned by the colony hybridization method or byculturing a variety of transformants in nutrient culture mediacontaining non-reducing saccharides having a trehalose structure as anend unit and having a degree of glucose polymerization of 3 or higher,and selecting the objective transformants which release trehalose formthe non-reducing saccharides.

The transformants thus obtained extracellularly produce the objectiveenzyme when cultured in nutrient culture media. Generally, liquid mediain general supplemented with carbon sources, nitrogen sources andminerals, and, if necessary, further supplemented with a small amount ofamino acids and vitamins can be used as the nutrient culture media.Examples of the carbon sources are saccharides such as starch, starchhydrolysate, glucose, fructose and sucrose. Examples of the nitrogensources are organic- and inorganic-substances containing nitrogen suchas ammonia, ammonium salts, urea, nitrate, peptone, yeast extract,defatted soy been, corn steep liquor and beef extract. Culturescontaining the objective enzyme can be prepared by inoculating thetransformants into nutrient culture media, and incubating them at atemperature of 25°-65° C. and a pH of 2-8 for about 1-6 days underaerobic aeration-agitation conditions. Such a culture can be used intactas an enzyme preparation, and, usually, it may be disrupted withultrasonic disintegrator and/or cell-wall lysis enzymes prior to use,followed by separating the enzyme from the intact cells and cell debrisby filtration and/or centrifugation, and purifying the enzyme. Themethods used for purifying the enzyme in the invention includeconventional ones in general. From cultures the intact cells and celldebris are eliminated and subjected to one or more methods such asconcentration, salting out, dialysis, separately sedimentation, gelfiltration chromatography, ion exchange chromatography, hydrophobicchromatography, affinity chromatography, gel electrophoresis andisoelectric point electrophoresis.

As is described above, the enzyme exerts a distinct activity of formingtrehalose from non-reducing saccharides having a trehalose structure asan end unit and having a degree of glucose polymerization of 3 orhigher, and such an activity has not yet been found in any conventionalenzymes. Therefore, the use of the enzyme facilitates the preparation oftrehalose in a relatively-high yield and efficiency from non-reducingsaccharides such as α-glucosyltrehalose, α-maltosyltrehalose,α-maltotriosyltrehalose, α-maltotetraosyltrehalose andα-maltopentaosyltrehalose in a considerably-high yield. Thesenon-reducing saccharides can be obtained in a satisfactorily-high yieldfrom starch hydrolysates, which are obtained by treating amylaceoussubstances such as starch, amylose and amylopectin prepared with acidsand/or amylases, by using non-reducing saccharide-forming enzyme asdisclosed in Japanese Patent Application No.349,216/93. Thus, trehalose,whose industrial preparation has been difficult, can be prepared fromstarch and amylaceous substances as a material with a relative easinessand in a desired amount when the present enzyme and the non-reducingsaccharide-forming enzyme, as disclosed in Japanese Patent ApplicationNo.349,216/93, are used in combination.

As described in "Handbook of Amylases and Related Enzymes", 1st edition,edited by The Amylase Research Society of Japan, published by PergamonPress plc, Oxford, England (1988), α-amylase, maltotetraose-formingamylase, maltopentaose-forming amylase and maltohexaose-forming amylaseare especially useful to prepare the reducing amylaceous saccharidesused in the invention, and, the use of any one of these amylases readilyyields amylaceous saccharide mixtures rich in reducing amylaceoussaccharides having a degree of glucose polymerization of 3 or higher ina considerably-high yield. If necessary, the combination use of such anamylase and a starch debranching enzyme such as pullulanase orisoamylase can increase the yield of the reducing amylaceous saccharidesusable as a substrate for the non-reducing saccharide-forming enzyme,i.e. the non-reducing saccharides can be obtained by coexisting thenon-reducing saccharide-forming enzyme in an aqueous solution containingas a substrate one or more of the reducing amylaceous saccharides in anamount up to a concentration of 50 w/v %, and subjecting the solution toan enzymatic reaction at a temperature of about 40-55° C. and a pH ofabout 6-8 until a desired amount of the objective non-reducingsaccharides are formed.

Usually, in the present conversion method, the recombinant enzymeaccording to the present invention is allowed to coexist in theaforesaid aqueous solution containing one or more of the non-reducingamylaceous saccharides, and to enzymatically react with the saccharideswhile keeping at a prescribed temperature and pH until a desired amountof trehalose is released.

Although the enzymatic reaction proceeds even below a concentration of0.1 w/v % of a substrate, a higher concentration of 2 w/v %, preferably,5-50 w/v % of a substrate can be satisfactorily used to apply thepresent conversion method to an industrial-scale production. Thetemperature and pH used in the enzymatic reaction are set within theranges of which do not inactivate the recombinant enzyme and allow therecombinant enzyme to effectively act on substrates, i.e. a temperatureup to about 55° C., preferably, a temperature in the range of about40°-55° C., and a pH of 5-10, preferably, a pH in the range of about6-8. The amount and reaction time of the present recombinant enzyme arechosen dependently on the enzymatic reaction conditions. The enzymaticreaction effectively converts non-reducing saccharides into saccharidecompositions containing trehalose and glucose and/ormaltooligosaccharides, and, in the case of using α-maltotriosyltrehaloseas a substrate, the conversion rate reaches to approximately 100%. Inthe case of simultaneously subjecting starch hydrolysates to the actionof either of the above amylases together with the non-reducingsaccharide-forming enzyme and the present recombinant enzyme,non-reducing saccharides are formed from the hydrolysates whilehydrolyzed into glucose and/or maltooligosaccharides, and because ofthis saccharide compositions with a relatively-high trehalose contentcan be effectively obtained in a relatively-high yield.

The reaction products obtained by the present conversion reaction can beused intact, and, usually, they are purified prior to use: Insolublesubstances are eliminated from the reaction products by filtration andcentrifugation, and the resultant solutions are decolored with activatedcharcoal, desalted and purified on ion exchangers, and concentrated intosyrupy products. Dependently on their use, the syrupy products are driedin vacuo and spray-dried into solid products. In order to obtainproducts which substantially consist of non-reducing saccharides, theabove mentioned syrupy products are subjected to one or more methodssuch as chromatography using an ion exchanger, activated charcoal andsilica gel to separate saccharides, separately sedimentation usingalcohol and/or acetone, membrane filtration, fermentation by yeasts, andremoval and decomposition of reducing saccharides by alkalis. Themethods to treat a large amount of reaction mixture are, for example,fixed bed- or pseudomoving bed-ion exchange column chromatography asdisclosed in Japanese Patent Laid-Open Nos.23,799/83 and 72,598/83, andsuch a method enables an effective industrial-scale production ofproducts with a relatively-high trehalose content.

These trehalose and compositions containing the same have a wideapplicability to a variety of products which are apt to be readilydamaged by the reducibility of saccharide sweeteners: For example, theycan be satisfactorily used as a sweetener, taste-improving agent,quality-improving agent, stabilizer, filler, excipient and adjuvant infood products in general, cosmetics and pharmaceuticals.

The following examples explain the present invention in more detail, andthe techniques themselves used in the examples are conventional ones inthis field, for example, those described by J. Sumbruck et al. in"Molecular Cloning A Laboratory Manual", 2nd edition, published by ColdSpring Harbor Laboratory Press (1989).

EXAMPLE 1 Preparation of recombinant DNA containing DNA encoding enzymeM-11 and transformant EXAMPLE 1-1 Preparation of chromosomal DNA

A seed culture of Rhizobium sp. M-11 was inoculated into bacto nutrientbroth medium (pH 7.0), and cultured at 27° C. for 24 hours with a rotaryshaker. The cells were separated from the resultant culture bycentrifugation, suspended in TES buffer (pH 8.0), admixed with 0.05 w/v% lysozyme, and incubated at 37° C. for 30 min. The resultant wasfreezed at -80° C. for one hour, admixed with TSS buffer (pH 9.0),heated to 60° C., and further admixed with a mixture solution of TESbuffer and phenol, and the resultant solution was chilled with ice,followed by centrifugally collecting the precipitated crude chromosomalDNA. To the supernatant was added 2 fold volumes of cold ethanol, andthe reprecipitated crude chromosomal DNA was collected, suspended in SSCbuffer (pH 7.1), admixed with 7.5 μg ribonuclease and 125 μg protease,and incubated at 37° C. for one hour. Thereafter, a mixture solution ofchloroform and isoamyl alcohol was added to the reaction mixture toextract the objective chromosomal DNA, and admixed with cold ethanol,followed by collecting the formed sediment containing the chromosomalDNA. The purified chromosomal DNA thus obtained was dissolved in SSCbuffer (pH 7.1) to give a concentration of about one mg/ml, and theresultant solution was freezed at -80° C.

EXAMPLE 1-2 Preparation of recombinant DNA pBMU27 and transformant BMU27

About one ml of the purified chromosomal DNA obtained in Example 1-1 wasplaced in a container, admixed with about 35 units of Sau 3AI, arestriction enzyme, and enzymatically reacted at 37° C. for about 20 minto partially digest the chromosomal DNA, followed by recovering a DNAfragment consisting of about 2,000-6,000 base pairs by means of sucrosedensity-gradient ultracentrifugation. One μg of Bluescript II SK(+), aplasmid vector, was provided, subjected to the action of Bam HI, arestriction enzyme, to completely digest the plasmid vector, admixedwith 10 μg of the DNA fragment and 2 units of T4 DNA ligase, and allowedto stand at 4° C. overnight to ligate the DNA fragment to the vectorfragment. To the resultant recombinant DNA was added 30 μl of "EpicurianColi® XLI-Blue", competent cell commercialized by Toyobo Co., Ltd.,Tokyo, Japan, allowed to stand under ice-chilling conditions for 30 min,heated to 42° C., admixed with SOC broth, and incubated at 37° C. forone hour to introduce the recombinant DNA into Escherichia coli.

The resultant transformant was inoculated into agar plate (pH 7.0)containing 50 μg/ml of 5-bromo-4-chloro-3-indolyl-β-galactoside, andcultured at 37° C. for 18 hours, followed by placing a nylon film on theagar plate to fix thereon about 6,000 colonies formed on the agar plate.Based on the amino acid sequence located at positions from 8 to 13 asshown in SEQ ID NO:7, i.e. Phe-Asp-Ile-Trp-Ala-Pro, the base sequence ofprobe 1 represented by 5'-TTYGAYATHTGGGCNCC-3'(SEQ ID NO:15) waschemically synthesized, labelled with ³² P, and hybridized with thecolonies of transformants fixed on the nylon film, followed by selecting14 transformants which exhibited a strong hybridization.

The objective recombinant DNA was selected in usual manner from the 14transformants, and, in accordance with the method described by E. M.Southern in Journal of Molecular Biology, Vol.98, pp.503-517 (1975), therecombinant DNA was hybridized with probe 2 having the base sequence asshown in SEQ ID NO:8, which had been chemically synthesized based on theamino acid sequence located at positions from 2 to 6, i.e.Asp-Trp-Ala-Glu-Ala, in SEQ ID NO:8, followed by selecting a recombinantDNA strongly hybridized with the probe 2. The recombinant DNA andtransformant thus selected were respectively named "pBMU27"and "BMU27".

The transformant BMU27 was inoculated into L-broth (pH 7.0) containing100 μg/ml ampicillin, and cultured at 37° C. for 24 hours by a rotaryshaker. After completion of the culture, the resultant cells werecollected from the culture by centrifugation, and treated with thealkaline method in general to extracellularly extract a recombinant DNA.The extract was in usual manner purified and analyzed to reveal that therecombinant DNA pBMU27 consists of about 5,700 base pairs and has thestructure expressed by the restriction map as shown in FIG. 9. It wasfound that, as shown in FIG. 9, the DNA which consists of 1,767 basepairs for encoding the enzyme M-11 is positioned in the downstream nearto the digested site of Eco RV, a restriction enzyme.

EXAMPLE 1-3 Production of enzyme by transformant BMU27

A liquid nutrient culture medium consisting of 2.0 w/v % "PINE-DEX #4",a starch hydrolysate commercialized by Matsutani Chemical Ind., Co.,Ltd., Tokyo, Japan, 0.5 w/v % peptone, 0.1 w/v % yeast extract, 0.1 w/v% disodium hydrogen phosphate and 0.1 w/v % potassium dihydrogenphosphate was adjusted to pH 7.0, admixed with 50 μg/ml ampicillin,autoclaved at 120° C. for 20 min, cooled and inoculated with a seedculture of transformant BMU27 obtained in Example 1-2, followed byculturing the transformant at 37° C. for 24 hours by a rotary shaker.The resultant culture was treated with ultrasonic disintegrator todisrupt cells, and the resultant suspension was centrifuged to removeinsoluble substances. The supernatant thus obtained was assayed for theenzyme activity to find that one L of the culture yielded about 4,000units of the enzyme.

As a control, a seed culture of Escherichia coli XLI-Blue or Rhizobiumsp. M-11 was inoculated in the same fresh preparation of the same liquidnutrient culture medium but free of ampicillin, and, in the case ofculturing Rhizobium sp. M-11, it was cultured and treated similarly asabove except that the cultivation temperature was set to 30° C. Assayingthe resultant activity, one L culture of Rhizobium sp. M-11 yieldedabout 2,000 units of the enzyme, and the yield was significantly lowerthan that of transformant BMU27. Escherichia coli XLI-Blue used as ahost did not form the enzyme.

Thereafter, the enzyme produced by the transformant MBU27 was purifiedsimilarly as in Experiment 1-1, and examined on the properties andcharacters. As a result, it was revealed that it has substantially thesame physicochemical properties as enzyme M-11, i.e. it has a molecularweight of about 57,000-68,000 daltons on SDS-PAGE and an isoelectricpoint of about 3.3-4.6 on isoelectrophoresis. The results indicate thatthe present enzyme can be prepared by the recombinant DNA technology,and the yield can be significantly increased thereby.

EXAMPLE 2 Preparation of complementary chain DNA derived from Rhizobiumsp. M-11 and determination for its base sequence and amino acid sequence

Two μg of the recombinant DNA pBMU27 obtained in Example 1-2 wasprovided, admixed with 2 M aqueous sodium hydroxide solution to effectdegeneration, and admixed with an adequate amount of cold ethanol,followed by collecting the formed sediment containing a template DNA anddrying the sediment in vacuo. To the template DNA were added 50 pmole/mlof a chemically synthesized primer 1 represented by5'-GTAAAACGACGGCCAGT-3'(SEQ ID NO:16), 10 μl of 40 mM Tris-HCl buffer(pH 7.5) containing 20 mM magnesium chloride and 20 mM sodium chloride,and the mixture was incubated at 65° C. for 2 min to effect annealingand admixed with 2 μl of an aqueous solution containing dATP, dGTP anddTTP in respective amounts of 7.5 μM, 0.5 μl of α-³² P!dCTP (2 mCi/ml),one μl of 0.1 M dithiothreitol, and 2 μl of 1. 5 units/ml T7 DNApolymerase, followed by incubating the resultant mixture at 25° C. for 5min to extend the primer 1 from the 5'-terminus to the 3'-terminus.Thus, a complementary chain DNA was formed.

The reaction product containing the complementary chain DNA was dividedinto quarters, to each of which 2.5 μl of 50 mM aqueous sodium chloridesolution containing 80 μM dNTP and 8 μM ddATP, ddCTP, ddGTP or ddTTP wasadded, and the resultant mixture was incubated at 37° C. for 5 min,followed by suspending the reaction by the addition of 4 μl of 98 v/v %aqueous formamide solution containing 20 mM EDTA, 0.05 w/v % bromophenolblue, and 0.05 w/v % xylene cyanol. The reaction mixture was heated witha boiling-water bath for 3 min, and a portion of which was placed on agel containing 6 w/v % polyacrylamide, and electrophoresed by energizingthe gel with a constant voltage of about 2,000 volts to separate DNAfragments, followed by fixing the gel in usual manner, drying the geland subjecting the resultant gel to autoradiography.

Analyses of the DNA fragments separated on the radiogram revealed thatthe complementary chain DNA contains the base sequence consisting ofabout 2,161 base pairs as shown in SEQ ID NO:11. An amino acid sequenceestimable from the base sequence was as shown in SEQ ID NO:12 and wascompared with the amino acid sequence containing the N-terminal or thepartial amino acid sequence of enzyme M-11 as shown in SEQ ID NO:5, 7 or8. As a result, it was found that the amino acid sequence containing theN-terminal of SEQ ID NO:5 corresponds to the amino acid sequence locatedat positions from 8 to 27 in SEQ ID NO:12, and the partial amino acidsequence of SEQ ID NO:7 or 8 corresponds to the amino acid sequencelocated at positions from 10 to 30 or at positions from 493 to 509 inSEQ ID NO:11. These results indicate that enzyme M-11 has the amino acidsequence of SEQ ID NO:2, and it is encoded by the DNA having the basesequence as shown in SEQ ID NO:1.

EXAMPLE 3 Preparation of recombinant DNA, containing DNA derived fromArthrobacter sp. 036, and transformant EXAMPLE 3-1 Preparation ofchromosomal DNA

Similarly as in Example 1-1, a chromosomal DNA was isolated fromArthrobacter sp. Q36, purified and dissolved in SSC buffer (pH 7.1) togive a concentration of about one mg/ml, and the resultant solution wasfreezed at -80° C. for storage.

EXAMPLE 3-2 Preparation of recombinant DNA pBRT32 and transformant BRT32

The purified chromosomal DNA obtained in Example 3-1 was partiallydigested similarly as in Example 1-2, followed by recovering a DNAfragment consisting of about 2,000-6,000 base pairs by sucrose densitygradient ultracentrifugation. The DNA fragment was ligated to a lysateof Bluescript II SK(+) which had been treated with Bam HI, and theresultant recombinant DNA was introduced into Escherichia coli XLI-Blue.The transformants thus obtained were cultured similarly as in Example1-2 on agar plates containing 5-bromo-4-chloro-3-indolyl-β-galactoside,and the formed about 5,000 colonies were fixed on a nylon film, whilethe probe 3 represented by 5'-ATGGGNTGGGAYCCNGC-3'was chemicallysynthesized based on the amino acid sequence of Met-Gly-Trp-Asp-Pro-Alalocated at positions from 5 to 10 in SEQ ID NO:9, labelled with ³² P,and hybridized with transformant colonies which had been fixed on thenylon film, followed by selecting 10 transformants which stronglyhybridized with the probe 3.

Similarly as in Example 1-2, the objective recombinant DNA was selectedfrom 10 transformants, and hybridized with probe 4 represented by5'-TAYGAYGTNTGGGC-3'(SEQ ID NO:18) which had been chemically synthesizedbased on the amino acid sequence of Tyr-Asp-Val-Trp-Ala located atpositions from 8 to 12 in SEQ ID NO:10, followed by selecting arecombinant DNA which strongly hybridized with probe 4. The recombinantDNA and transformant thus selected were respectively named "pBRT32"and"BRT32".

The transformant BRT32 was inoculated into L-broth containingampicillin, and cultured similarly as in Example 1-2, and theproliferated cells were collected from the resultant culture, and fromwhich a recombinant DNA was extracted, purified and analyzed to revealthat the recombinant DNA pBRT32 consists of about 6,200 base pairs andhas the structure of the restriction map as shown in FIG. 10. As shownin FIG. 10, it was revealed that the DNA, which consists of 1,791 basepairs for encoding the DNA of enzyme Q36, is located in the downstreamnear to the cleavage site of Kpn I.

EXAMPLE 3-3 Production of enzyme by transformant BRT32

A liquid nutrient culture medium consisting of 2.0 w/v % "PINE-DEX #4",a starch hydrolysate commercialized by Matsutani Chemical Ind., Co.,Ltd., Tokyo, 0.5 w/v % peptone, 0.1 w/v % yeast extract, 0.1 w/v %disodium hydrogen phosphate and 0.1 w/v % potassium dihydrogen phosphatewas adjusted to pH 7.0, admixed with 50 μg/ml ampicillin, autoclaved at120° C. for 20 min, cooled and inoculated with a seed culture of thetransformant BRT32 obtained in Example 3-2, followed by culturing thetransformant at 37° C. for 24 hours by a rotary shaker. The resultantculture was treated with an ultrasonic disintegrator to disrupt cells,and the resultant suspension was centrifuged to remove insolublesubstances. The supernatant thus obtained was assayed for the presentenzyme activity to find that one L of the culture yielded about 3,900units of the enzyme.

As a control, a seed culture of Escherichia coli XLI-Blue orArthrobacter sp. Q36 was inoculated into a fresh preparation of the sameliquid nutrient culture medium but free of ampicillin, and, in the caseof culturing Arthrobacter sp. Q36, it was cultured and treated similarlyas above except that the cultivation temperature was set to 30° C.Assaying the enzyme activity, one L of the culture of Arthrobacter sp.Q36 yielded about 1,800 units of the enzyme, and the yield wassignificantly lower than that of the transformant BRT32. The Escherichiacoli XLI-Blue used as a host did not form the enzyme.

Thereafter, the enzyme produced by the transformant BRT32 was purifiedsimilarly as in Experiment 1-1, and examined on the properties andcharacters to reveal that it has substantially the same physicochemicalproperties as that of enzyme Q36, i.e. it has a molecular weight ofabout 57,000-68,000 daltons on SDS-PAGE and an isoelectric point ofabout 3.3-4.6 on isoelectrophoresis. These results indicate that theenzyme can be prepared by the recombinant DNA technology, and the yieldcan be significantly increased thereby.

EXAMPLE 4 Preparation of complementary chain DNA derived fromArthrobacter sp. 036, and determination for its base sequence and aminoacid sequence

The recombinant DNA pBRT32 obtained in Example 3-2 was similarly treatedas in Example 2 to form a template DNA which was then annealed togetherwith the primer 1, followed by allowing T7 DNA polymerase to act on theresultant to extend the primer 1 from the 5'-terminus to the 3'-terminusto obtain a complementary chain DNA. Similarly as in Example 2, thecomplementary chain DNA was subjected to the dideoxy chain terminatormethod to analyze DNA fragments which had been isolated on a radiogram.The result revealed that the complementary chain DNA contained a basesequence consisting of 2,056 base pairs as shown in SEQ ID NO:12. Anamino acid sequence estimable from the base sequence was as shown in SEQID NO:14, and compared with the amino acid sequence containing theN-terminal or the partial amino acid sequence of SEQ ID NO:6, 9 or 10.As a result, it was found that the amino acid sequence of SEQ ID NO:6corresponds to that located at positions from 2 to 21 in SEQ ID NO:12,and that the partial amino acid sequence in SEQ ID NO:9 or 10corresponds to that located at positions from 470 to 489 or at positionsfrom 12 to 31 in SEQ ID NO:14. These results indicate that enzyme Q36has the amino acid sequence of SEQ ID NO:4, and it is encoded by the DNAhaving the base sequence as shown in SEQ ID NO:3.

EXAMPLE 5 Preparation of recombinant enzyme

In 500-ml Erlenmeyer flasks were placed 100 ml aliquots of a liquidnutrient culture medium (pH 7.0) consisting of 2.0 w/v % "PINE-DEX #4",a starch hydrolysate commercialized by Matsutani Chemical Ind., Co.,Ltd., Tokyo, Japan, 0.5 w/v % peptone, 0.1 w/v % yeast extract, 0.1 w/v% disodium hydrogen phosphate and 0.1 w/v % potassium dihydrogenphosphate, and to each flask was added 50 μg/ml ampicillin andautoclaved at 120° C. for 20 min. Thereafter, the flasks were cooled andinoculated with a seed culture of the transformant BMU27 obtained inExample 1-2, followed by culturing the transformant at 27° C. for 24hours by a rotary shaker. Apart from this, 18 L of a fresh preparationof the same liquid culture medium was placed in a 30-L jar fermentor,admixed with 50 μg/ml ampicillin, sterilized at 120° C. for 20 min,cooled and inoculated with one v/v % of the seed culture obtained in theabove, followed by the culture at 37° C. for 24 hours while keeping thepH at 6-8 under aeration-agitation conditions. The resultant culture wastreated with an ultrasonic disintegrator to disrupt cells, and theresultant suspension was centrifuged to remove insoluble substances. Thesupernatant thus obtained was assayed for the enzyme activity to revealthat one L of the culture yielded about 3,900 units of the enzyme. Thesupernatant was purified by the method in Experiment 1-1 to obtain anabout 67 ml aqueous solution containing an about 165 units/ml of arecombinant enzyme having a specific activity of about 290 units/mgprotein.

EXAMPLE 6 Preparation of recombinant enzyme

Recombinant BRT32 obtained by the method in Experiment 3-2 was culturedsimilarly as in Example 5, and the resultant culture was treated with anultrasonic integrator to disrupt cells. The resultant suspension wascentrifuged to remove insoluble substances, and the resultantsupernatant was assayed for the enzyme activity to have an activity ofabout 4,000 units per L. The supernatant was purified by the method inExperiment 1-1 to obtain an about 55 ml aqueous solution containingabout 200 units/ml of a recombinant enzyme with a specific activity ofabout 420 units/mg protein.

EXAMPLE 7 Conversion of non-reducing saccharide by recombinant enzymeEXAMPLE 7-1 (a) Preparation of non-reducing saccharide-forming enzyme

To 500-ml Erlenmeyer flasks were placed 100 ml aliquots of a liquidnutrient culture medium (pH 7.0) consisting of 2.0 w/v % maltose, 0.5w/v % peptone, 0.1 w/v % yeast extract, 0.1 w/v % disodium hydrogenphosphate and 0.1 w/v % potassium dihydrogen phosphate, and the flaskswere autoclaved at 120° C. for 20 min. Thereafter, the flasks werecooled and inoculated with a seed culture of Rhizobium sp. M-11,followed by culturing it at 27° C. for 24 hours by a rotary shaker.Apart from this, 20 L of a fresh preparation of the same liquid culturemedium was placed in a 30-L jar fermentor, and sterilized, inoculatedwith one v/v % of the seed culture obtained in the above, followed bythe culture at 30° C. and at a pH of 7-8 for 24 hours underaeration-agitation conditions. Thereafter, the resultant culture wastreated with an ultrasonic disintegrator to disrupt cells, and theresultant suspension was centrifuged to remove insoluble substances andpurified according to the method in Experiment 1-1 to obtain anon-reducing saccharide-forming enzyme having a specific activity ofabout 195 units/mg protein in a yield of about 220 units per L of theculture.

Throughout the specification the activity of a non-reducingsaccharide-forming enzyme is expressed by the value measured on thefollowing assay: Place 4 ml of 50 mM phosphate buffer (pH 7.0)containing 1.25 w/v % maltopentaose in a test tube, add one ml of anenzyme solution to the test tube, and incubate the solution at 40° C.for 60 min to effect enzymatic reaction. Thereafter, the reactionmixture is heated at 100° C. for 10 min to suspend the enzymaticreaction, followed by diluting it with distilled water by 10 times andassaying the reducing activity by the Somogyi-Nelson's method. One unitactivity of the non-reducing saccharide-forming enzyme is defined as theamount of enzyme which decreases the reducing power corresponding to oneμmol maltopentaose per min under the above conditions.

EXAMPLE 7-1(b) Preparation of syrupy product containing trehalose

A potato starch was suspended in water to give a 15 w/w % suspensionwhich was then mixed with 0.1 w/w % calcium carbonate. The mixture wasadjusted its pH to 6.0, mixed with 0.2 w/w %, d.s.b., of "TERMAMYL 60L",an α-amylase specimen commercialized by Novo Nordisk Bioindustri A/S,Copenhagen, Denmark, and enzymatically reacted at 95° C. for 15 min toeffect gelatinization and liquefaction. The liquefied solution wasautoclaved at 120° C. for 30 min to inactivate the remaining enzyme,rapidly cooled to 45° C., 1,000 units/g starch, d.s.b., of pullulanasecommercialized by Hayashibara Biochemical Laboratories., Inc., Okayama,Japan, 3.4 units/g starch, d.s.b., of the non-reducingsaccharide-forming enzyme obtained in Example 7-1(a), and 4.2 units/gstarch, d.s.b., of the recombinant enzyme obtained by the method inExample 5, followed the enzymatic reaction for 48 hours. The reactionmixture was heated at 95° C. for 10 min to inactivate the remainingenzyme, cooled, filtered, and, in usual manner, decolored with anactivated charcoal, desalted and purified with an ion-exchange resin,and concentrated to obtain a syrupy product with a concentration ofabout 60 w/w % in a yield of about 92%, d.s.b.

Analysis of the syrup by the method of Experiment 2-1 revealed that itcontained 70.2 w/w % trehalose, 2.4 w/w % α-glucosyltrehalose, 3.3 w/w %α-maltosyltrehalose, 0.7 w/w % glucose, 10.1 w/w % maltose, 12.9 w/w %maltotriose, and 0.4 w/w % maltooligosaccharides having a degree ofglucose polymerization of 4 or higher. The product, having a mild andmoderate sweetness as well as an adequate viscosity andmoisture-retaining ability, can be satisfactorily used in food productsin general, cosmetics and pharmaceuticals as a sweetener,taste-improving agent, quality-improving agent, stabilizer, filler,excipient and adjuvant.

EXAMPLE 7-1(c) Preparation of Powdery product containing trehalose

To 4 jacketed-stainless steel columns, having a diameter of 5.4 cm and alength of 5 m each was packed homogeneity with "XT-1016 (Na⁺ -form)", astrong-acid cation exchange resin commercialized by Tokyo OrganicChemical Industries, Ltd., Tokyo, Japan, and the columns were cascadedin series to give a total column length of 20 m. The syrupy productobtained in Example 7-1(b) was fed to the columns at a rate of about 5v/v % against the resin at an inner column temperature of 55° C., andthe columns were fed with 55° C. hot water at an SV (space velocity )0.3 to fractionate saccharides in the syrupy product. Based on theanalysis of the saccharide composition of the eluate, fractions rich intrehalose were collected, pooled, concentrated, dried in vacuo andpulverized to obtain a solid product containing about 97 w/w % trehalosein a yield of about 56% against the starting material, d.s.b.

The product, having a mild sweetness and substantially free ofreducibility, can be satisfactorily used in food products in general,cosmetics and pharmaceuticals as a sweetener, taste-improving agent,quality-improving agent, stabilizer, filler, excipient and adjuvant.

EXAMPLE 7-1(d) Preparation of powdery crystalline trehalose

A portion of the trehalose rich fraction obtained in Example 7-1(c) wasconcentrated into an about 75 w/w % solution which was then transferredto a crystallizer, admixed with about 2 w/w %, d.s.b., hydrouscrystalline trehalose as a seed crystal, and crystallized under gentlestirring conditions to obtain a massecuite with a crystallinity of about45 w/w %. The massecuite was sprayed downward from a nozzle, equipped atthe upper part of a spraying tower at a pressure of about 150 kg/cm²while about 85° C. hot air was flowing downward from the upper part ofthe tower to accumulate a crystalline powder on a belt conveyer providedon the basement of the tower, followed by gradually transferring it outof the tower. Thereafter, the powder was transferred to an ageing towerand aged for 10 hours to complete the crystallization and drying whilean about 40° C. hot air was blowing to the contents. Thus, a powderyproduct containing hydrous crystalline trehalose was obtained in a yieldof about 90 w/w % against the starting material, d.s.b.

The product, having a substantial non-hygroscopicity and a mild andhigh-quality sweetness, can be satisfactorily used in food products ingeneral, cosmetics, pharmaceuticals and feeds as a sweetener,taste-improving agent, quality-improving agent, stabilizer, filler,excipient and adjuvant.

EXAMPLE 8 Conversion of non-reducinq saccharide by recombinant enzyme

Potato starch was suspended in water to give a concentration of 6 w/w %,d.s.b., and the suspension was admixed with 500 units/g starch ofisoamylase commercialized by Hayashibara Biochemical Laboratories,.Inc., Okayama, Japan, and enzymatically reacted for 20 hours. Thereaction mixture was adjusted to a pH of 6.5, autoclaved at 120° C. for10 min to inactivate the remaining enzyme, rapidly cooled to 95° C.,admixed with 0.1 w/w t per g starch, d.s.b., of "TERMAMYL 60L", anα-amylase specimen commercialized by Novo Nordisk Bioindustri A/S,Copenhagen, Denmark, and enzymatically reacted for 15 min. The reactionmixture was heated at 130° C. for 30 min to inactivate the remainingenzyme, rapidly cooled to 45° C., admixed with 4.1 units/g starch,d.s.b., of a non-reducing saccharide-forming enzyme obtained by themethod in Example 7-1(a), and 4.9 units/g starch, d.s.b., of the presentrecombinant enzyme obtained by the method in Example 6, andenzymatically reacted for 64 hours. The reaction mixture was heated at95° C. for 10 min to inactivate the remaining enzyme, rapidly cooled to55° C., adjusted to pH 5.0, admixed with 10 units/g starch, d.s.b., of"GLUCOZYME", a glucoamylase specimen commercialized by NagaseBiochemicals, Ltd., Kyoto, Japan, and enzymatically reacted for 40hours. The reaction mixture was heated at 95° C. for 10 min toinactivate the remaining enzyme, cooled, filtered, and, in usual manner,decolored with an activated charcoal, desalted and purified with anion-exchange resin, and concentrated to obtain an about 60 w/w % syrupyproduct containing about 80.5 w/w % trehalose, d.s.b. The syrupy productwas concentrated into an about 84 w/w % syrup which was then transferredto a crystallizer, admixed with an about 2 w/w % hydrous crystallinetrehalose, d.s.b., and crystallized under gentle stirring conditions toobtain a massecuite having a crystallinity of about 45 w/w %. Themassecuite was distributed to plastic plain vessels which were thenallowed to stand at ambient temperature for 3 days to effectsolidification and aging, followed by detaching the resultant blocksfrom the vessels and pulverizing the blocks with a cutter to obtain asolid product containing hydrous crystalline trehalose in a yield ofabout 90 w/w % against the material starch, d.s.b.

The product, which is substantially free of hygroscopicity and readilyhandleable, can be arbitrarily used in food products in general,cosmetics, pharmaceuticals as a sweetening agent, taste-improving agent,quality-improving agent, stabilizer, filler, excipient and adjuvant.

EXAMPLE 9 Conversion of non-reducing saccharide by recombinant enzyme

Potato starch was suspended in water to give a concentration of 6 w/w %,d.s.b., and the suspension was admixed with 0.01 w/w % "NEO-SPITASE",a-amylase commercialized by Nagase Biochemicals, Ltd., Kyoto, Japan,adjusted to pH 6.2, and enzymatically reacted at 85°-90° C. for 20 minto gelatinize and liquefy the starch. The liquefied starch was heated at120° C. for 10 min to inactivate the remaining enzyme, rapidly cooled to45° C., admixed with 500 units/g starch, d.s.b., of isoamylasecommercialized by Hayashibara Biochemical Laboratories, Inc., Okayama,Japan, 3.2 units/g starch, d.s.b., of a non-reducing saccharide-formingenzyme obtained by the method in Example 7-1(a), and 5.0 units/g starch,d.s.b., of the present recombinant enzyme obtained by the method inExample 5, and enzymatically reacted for 48 hours. The reaction mixturewas heated at 95° C. for 10 min to inactivate the remaining enzyme,rapidly cooled to 55° C., adjusted to pH 5.0, admixed with 10 units/gstarch, d.s.b., of "GL-UCOZYME", glucoamylase commercialized by NagaseBiochemicals Ltd., Kyoto, Japan, and enzymatically reacted for 40 hours.The reaction mixture was heated at 95° C. for 10 min to inactivate theremaining enzyme, rapidly cooled, filtered, and, in usual manner,decolored with an activated charcoal, desalted and purified with anion-exchange resin, and concentrated to give a concentration of about 60w/w %, d.s.b., to obtain a syrupy product containing 78.3 w/w %trehalose, d.s.b. The syrupy product was fractionated similarly as inExample 7-1(c) except for using "CG6000(Na⁺)", a strong-acid cationexchange resin commercialized by Japan Organo, Co., Ltd., Tokyo, Japan,to obtain a fraction containing abut 95 w/w % trehalose, d.s.b. Thefraction was concentrated to give a concentration of about 75 w/w %,d.s.b., and, similarly as in Example 8, crystallized, and the resultantmassecuite in the form of block was pulverized to obtain a powderyproduct containing hydrous crystalline trehalose in a yield of about 70w/w % against the material starch, d.s.b.

The product, which is substantially free of hygroscopicity and readilyhandleable, can be arbitrarily used in food products in general,cosmetics, pharmaceuticals as a sweetening agent, taste-improving agent,quality-improving agent, stabilizer, filler, excipient and adjuvant.

As is described above, the present invention is based on the findingthat a novel enzyme which releases trehalose from non-reducingsaccharides having a trehalose structure as an end unit and having adegree of glucose polymerization of 3 or higher. The present inventionis to explore a way to produce the enzyme in a relatively-large scaleand in a considerably-high yield. The enzyme produced by thetransformant according to the present invention is the one characterizedby its revealed total amino acid sequence, and because of this it can beused for the preparations of trehalose which is premised on being usedin food products without fear of causing side effects.

Therefore, the present invention is an useful invention which exerts theaforesaid significant action and effect as well as giving a greatcontribution to this field.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 18                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1767 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..1767                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       GCCAAGCCGGTGCAGGGAGCGGGGCGCTTCGATATCTGGGCGCCCGAG48                            AlaLysProValGlnGlyAlaGlyArgPheAspIleTrpAlaProGlu                              151015                                                                        GCAGGCACCGTAACGCTGCTGGCCGGCGGGGAGCGCTACGAGATGGGC96                            AlaGlyThrValThrLeuLeuAlaGlyGlyGluArgTyrGluMetGly                              202530                                                                        CGCCGCCCCGGCAACGGGCCGGCGGACGAAGGCTGGTGGACGGCCGCG144                           ArgArgProGlyAsnGlyProAlaAspGluGlyTrpTrpThrAlaAla                              354045                                                                        GATGCACCGACAGGCGCGGACGTGGACTACGGATACCTGCTCGACGGC192                           AspAlaProThrGlyAlaAspValAspTyrGlyTyrLeuLeuAspGly                              505560                                                                        GACGAAATCCCGCTGCCGGACCCCCGGACCCGCCGCCAGCCCGAAGGC240                           AspGluIleProLeuProAspProArgThrArgArgGlnProGluGly                              65707580                                                                      GTCCATGCCCTGTCCCGGACCTTCGACCCCGGCGCCCACCGCTGGCAG288                           ValHisAlaLeuSerArgThrPheAspProGlyAlaHisArgTrpGln                              859095                                                                        GACGCCGGGTGGCAGGGCAGGGAACTCCAGGGCTCCGTGATTTACGAA336                           AspAlaGlyTrpGlnGlyArgGluLeuGlnGlySerValIleTyrGlu                              100105110                                                                     CTCCACATCGGAACGTTCACGCCGGAAGGGACGCTGGACGCCGCCGCG384                           LeuHisIleGlyThrPheThrProGluGlyThrLeuAspAlaAlaAla                              115120125                                                                     GGCAAGCTGGACTACCTCGCCGGCCTGGGCATCGACTTCATTGAGCTG432                           GlyLysLeuAspTyrLeuAlaGlyLeuGlyIleAspPheIleGluLeu                              130135140                                                                     CTGCCCGTGAATGCCTTCAACGGCACGCACAACTGGGGCTACGACGGC480                           LeuProValAsnAlaPheAsnGlyThrHisAsnTrpGlyTyrAspGly                              145150155160                                                                  GTCCAGTGGTTTGCCGTGCATGAAGGCTACGGCGGGCCTGCGGCGTAC528                           ValGlnTrpPheAlaValHisGluGlyTyrGlyGlyProAlaAlaTyr                              165170175                                                                     CAGCGGTTCGTGGATGCGGCCCACGCGGCCGGCCTCGGCGTCATCCAG576                           GlnArgPheValAspAlaAlaHisAlaAlaGlyLeuGlyValIleGln                              180185190                                                                     GACGTGGTCTACAACCACCTCGGGCCGAGCGGGAACTACCTCCCCAGG624                           AspValValTyrAsnHisLeuGlyProSerGlyAsnTyrLeuProArg                              195200205                                                                     TACGGCCCGTACCTCAAGCACGGCGAAGGCAACACCTGGGGCGATTCG672                           TyrGlyProTyrLeuLysHisGlyGluGlyAsnThrTrpGlyAspSer                              210215220                                                                     GTCAACCTGGACGGGCCGGGATCCGACCACGTCCGCCAGTACATCCTG720                           ValAsnLeuAspGlyProGlySerAspHisValArgGlnTyrIleLeu                              225230235240                                                                  GACAACGTGGCCATGTGGCTGCGCGACTACCGGGTGGACGGCCTCCGC768                           AspAsnValAlaMetTrpLeuArgAspTyrArgValAspGlyLeuArg                              245250255                                                                     CTGGACGCCGTCCACGCCCTGAAGGATGAGCGGGCCGTCCACATCCTG816                           LeuAspAlaValHisAlaLeuLysAspGluArgAlaValHisIleLeu                              260265270                                                                     GAGGAGTTCGGCGCGCTGGCGGACGCCCTGTCGTCCGAAGGCGGCCGC864                           GluGluPheGlyAlaLeuAlaAspAlaLeuSerSerGluGlyGlyArg                              275280285                                                                     CCGCTGACCCTCATCGCCGAGTCCGACCTCAACAATCCGCGGCTGCTG912                           ProLeuThrLeuIleAlaGluSerAspLeuAsnAsnProArgLeuLeu                              290295300                                                                     TACCCCCGGGATGTCAACGGCTACGGACTGGCCGGCCAGTGGAGCGAC960                           TyrProArgAspValAsnGlyTyrGlyLeuAlaGlyGlnTrpSerAsp                              305310315320                                                                  GACTTCCACCACGCCGTGCACGTCAACGTCAGCGGGGAAACCACCGGC1008                          AspPheHisHisAlaValHisValAsnValSerGlyGluThrThrGly                              325330335                                                                     TACTACAGCGACTTCGACTCGCTCGGAGCCCTCGCCAAGGTCCTGCGT1056                          TyrTyrSerAspPheAspSerLeuGlyAlaLeuAlaLysValLeuArg                              340345350                                                                     GACGGGTTCTTCCACGACGGCAGCTACTCCAGCTTCCGCGGCCGCTGC1104                          AspGlyPhePheHisAspGlySerTyrSerSerPheArgGlyArgCys                              355360365                                                                     CACGGCCGGCCGATCAACTTCAGCGCCGTGCATCCGGCCGCGCTGGTG1152                          HisGlyArgProIleAsnPheSerAlaValHisProAlaAlaLeuVal                              370375380                                                                     GTCTGCTCACAGAACCATGACCAGATCGGCAACCGGGCCACCGGGGAC1200                          ValCysSerGlnAsnHisAspGlnIleGlyAsnArgAlaThrGlyAsp                              385390395400                                                                  CGGCTGTCCCAGTCACTTCCGTACGGCAGCCTGGCCCTGGCCGCCGTG1248                          ArgLeuSerGlnSerLeuProTyrGlySerLeuAlaLeuAlaAlaVal                              405410415                                                                     CTGACCCTCACCGGTCCGTTCACGCCCATGCTGTTCATGGGAGAGGAA1296                          LeuThrLeuThrGlyProPheThrProMetLeuPheMetGlyGluGlu                              420425430                                                                     TACGGGGCCACCACCCCGTGGCAGTTCTTCACCTCGCACCCTGAACCC1344                          TyrGlyAlaThrThrProTrpGlnPhePheThrSerHisProGluPro                              435440445                                                                     GAGCTGGGCAAGGCCACGGCCGAGGGCAGGATCAGGGAGTTCGAGCGC1392                          GluLeuGlyLysAlaThrAlaGluGlyArgIleArgGluPheGluArg                              450455460                                                                     ATGGGGTGGGATCCCGCCGTCGTGCCCGATCCGCAGGATCCGGAGACC1440                          MetGlyTrpAspProAlaValValProAspProGlnAspProGluThr                              465470475480                                                                  TTCACCCGCTCCAAACTGGACTGGGCGGAAGCGTCCGCCGGCGATCAT1488                          PheThrArgSerLysLeuAspTrpAlaGluAlaSerAlaGlyAspHis                              485490495                                                                     GCCCGCCTCCTGGAGCTGTACCGCTCGCTTATCACGCTGCGGCGGTCA1536                          AlaArgLeuLeuGluLeuTyrArgSerLeuIleThrLeuArgArgSer                              500505510                                                                     ACTCCGGAGCTCGCGCGCCTGGGCTTTGCGGACACCGCCGTCGAGTTC1584                          ThrProGluLeuAlaArgLeuGlyPheAlaAspThrAlaValGluPhe                              515520525                                                                     GACGACGACGCCCGCTGGCTCCGTTATTGGCGCGGAGGCGTGCAGGTG1632                          AspAspAspAlaArgTrpLeuArgTyrTrpArgGlyGlyValGlnVal                              530535540                                                                     GTGCTGAACTTCGCGGACCGTCCCATCAGCCTGGACCGGCCGGGAACC1680                          ValLeuAsnPheAlaAspArgProIleSerLeuAspArgProGlyThr                              545550555560                                                                  GCGCTGCTGCTCGCCACCGACGACGCCGTCCGGATGGACGGAGTCCAG1728                          AlaLeuLeuLeuAlaThrAspAspAlaValArgMetAspGlyValGln                              565570575                                                                     GTGGAGCTGCCGCCGCTGAGCGCCGCGGTTCTGCGCGAC1767                                   ValGluLeuProProLeuSerAlaAlaValLeuArgAsp                                       580585                                                                        (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 589 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       AlaLysProValGlnGlyAlaGlyArgPheAspIleTrpAlaProGlu                              151015                                                                        AlaGlyThrValThrLeuLeuAlaGlyGlyGluArgTyrGluMetGly                              202530                                                                        ArgArgProGlyAsnGlyProAlaAspGluGlyTrpTrpThrAlaAla                              354045                                                                        AspAlaProThrGlyAlaAspValAspTyrGlyTyrLeuLeuAspGly                              505560                                                                        AspGluIleProLeuProAspProArgThrArgArgGlnProGluGly                              65707580                                                                      ValHisAlaLeuSerArgThrPheAspProGlyAlaHisArgTrpGln                              859095                                                                        AspAlaGlyTrpGlnGlyArgGluLeuGlnGlySerValIleTyrGlu                              100105110                                                                     LeuHisIleGlyThrPheThrProGluGlyThrLeuAspAlaAlaAla                              115120125                                                                     GlyLysLeuAspTyrLeuAlaGlyLeuGlyIleAspPheIleGluLeu                              130135140                                                                     LeuProValAsnAlaPheAsnGlyThrHisAsnTrpGlyTyrAspGly                              145150155160                                                                  ValGlnTrpPheAlaValHisGluGlyTyrGlyGlyProAlaAlaTyr                              165170175                                                                     GlnArgPheValAspAlaAlaHisAlaAlaGlyLeuGlyValIleGln                              180185190                                                                     AspValValTyrAsnHisLeuGlyProSerGlyAsnTyrLeuProArg                              195200205                                                                     TyrGlyProTyrLeuLysHisGlyGluGlyAsnThrTrpGlyAspSer                              210215220                                                                     ValAsnLeuAspGlyProGlySerAspHisValArgGlnTyrIleLeu                              225230235240                                                                  AspAsnValAlaMetTrpLeuArgAspTyrArgValAspGlyLeuArg                              245250255                                                                     LeuAspAlaValHisAlaLeuLysAspGluArgAlaValHisIleLeu                              260265270                                                                     GluGluPheGlyAlaLeuAlaAspAlaLeuSerSerGluGlyGlyArg                              275280285                                                                     ProLeuThrLeuIleAlaGluSerAspLeuAsnAsnProArgLeuLeu                              290295300                                                                     TyrProArgAspValAsnGlyTyrGlyLeuAlaGlyGlnTrpSerAsp                              305310315320                                                                  AspPheHisHisAlaValHisValAsnValSerGlyGluThrThrGly                              325330335                                                                     TyrTyrSerAspPheAspSerLeuGlyAlaLeuAlaLysValLeuArg                              340345350                                                                     AspGlyPhePheHisAspGlySerTyrSerSerPheArgGlyArgCys                              355360365                                                                     HisGlyArgProIleAsnPheSerAlaValHisProAlaAlaLeuVal                              370375380                                                                     ValCysSerGlnAsnHisAspGlnIleGlyAsnArgAlaThrGlyAsp                              385390395400                                                                  ArgLeuSerGlnSerLeuProTyrGlySerLeuAlaLeuAlaAlaVal                              405410415                                                                     LeuThrLeuThrGlyProPheThrProMetLeuPheMetGlyGluGlu                              420425430                                                                     TyrGlyAlaThrThrProTrpGlnPhePheThrSerHisProGluPro                              435440445                                                                     GluLeuGlyLysAlaThrAlaGluGlyArgIleArgGluPheGluArg                              450455460                                                                     MetGlyTrpAspProAlaValValProAspProGlnAspProGluThr                              465470475480                                                                  PheThrArgSerLysLeuAspTrpAlaGluAlaSerAlaGlyAspHis                              485490495                                                                     AlaArgLeuLeuGluLeuTyrArgSerLeuIleThrLeuArgArgSer                              500505510                                                                     ThrProGluLeuAlaArgLeuGlyPheAlaAspThrAlaValGluPhe                              515520525                                                                     AspAspAspAlaArgTrpLeuArgTyrTrpArgGlyGlyValGlnVal                              530535540                                                                     ValLeuAsnPheAlaAspArgProIleSerLeuAspArgProGlyThr                              545550555560                                                                  AlaLeuLeuLeuAlaThrAspAspAlaValArgMetAspGlyValGln                              565570575                                                                     ValGluLeuProProLeuSerAlaAlaValLeuArgAsp                                       580585                                                                        (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1791 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..1791                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       ACGCACACCTACCCGCGGGAAGCCGCGAAACCCGTCCTGGGCCCCGCA48                            ThrHisThrTyrProArgGluAlaAlaLysProValLeuGlyProAla                              590595600605                                                                  CGCTACGACGTCTGGGCGCCCAACGCTGAATCCGTGACGCTGCTGGCC96                            ArgTyrAspValTrpAlaProAsnAlaGluSerValThrLeuLeuAla                              610615620                                                                     GGCGGGGAGCGCTACGCCATGCAGCGCCGGGCCGAGACCGGGCCGGAG144                           GlyGlyGluArgTyrAlaMetGlnArgArgAlaGluThrGlyProGlu                              625630635                                                                     GACGCCGGCTGGTGGACCGCCGCCGGCGCGCCTACGGATGGCAACGTG192                           AspAlaGlyTrpTrpThrAlaAlaGlyAlaProThrAspGlyAsnVal                              640645650                                                                     GACTACGGGTACCTTCTGGACGGCGACGAAACACCGCTTCCGGATCCA240                           AspTyrGlyTyrLeuLeuAspGlyAspGluThrProLeuProAspPro                              655660665                                                                     CGGACCCGCCGCCAGCCCGACGGCGTCCACGCCCTGTCCCGCACGTTC288                           ArgThrArgArgGlnProAspGlyValHisAlaLeuSerArgThrPhe                              670675680685                                                                  GACCCGTCCGCGTACAGCTGGCAGGACGACGCCTGGCAGGGCAGGGAA336                           AspProSerAlaTyrSerTrpGlnAspAspAlaTrpGlnGlyArgGlu                              690695700                                                                     CTGCAGGGCGCCGTCATCTACGAGCTCCACCTCGGAACATTCACGCCC384                           LeuGlnGlyAlaValIleTyrGluLeuHisLeuGlyThrPheThrPro                              705710715                                                                     GAAGGGACGCTGGAGGCGGCCGCCGGAAAGCTGGACTACCTCGCCGGC432                           GluGlyThrLeuGluAlaAlaAlaGlyLysLeuAspTyrLeuAlaGly                              720725730                                                                     TTGGGCGTCGACTTCATCGAGCTGCTGCCGGTGAACGCTTTCAACGGC480                           LeuGlyValAspPheIleGluLeuLeuProValAsnAlaPheAsnGly                              735740745                                                                     ACGCACAACTGGGGTTACGACGGTGTCCAGTGGTTCGCTGTGCACGAG528                           ThrHisAsnTrpGlyTyrAspGlyValGlnTrpPheAlaValHisGlu                              750755760765                                                                  GCATACGGCGGGCCGGAAGCGTACCAGCGGTTCGTCGACGCCGCCCAC576                           AlaTyrGlyGlyProGluAlaTyrGlnArgPheValAspAlaAlaHis                              770775780                                                                     GCCGCAGGCCTTGGCGTGATCCAGGACGTGGTCTACAACCACCTCGGC624                           AlaAlaGlyLeuGlyValIleGlnAspValValTyrAsnHisLeuGly                              785790795                                                                     CCCAGCGGGAACTACCTGCCGCGGTTCGGGCCGTACCTCAAGCAGGGC672                           ProSerGlyAsnTyrLeuProArgPheGlyProTyrLeuLysGlnGly                              800805810                                                                     GAGGGTAACACGTGGGGCGACTCGGTGAACCTGGACGGGCCCGGCTCC720                           GluGlyAsnThrTrpGlyAspSerValAsnLeuAspGlyProGlySer                              815820825                                                                     GACCATGTGCGCCGGTACATCCTGGACAACCTGGCCATGTGGCTGCGT768                           AspHisValArgArgTyrIleLeuAspAsnLeuAlaMetTrpLeuArg                              830835840845                                                                  GACTACCGGGTGGACGGCCTGCGGCTGGACGCCGTCCACGCCCTGAAG816                           AspTyrArgValAspGlyLeuArgLeuAspAlaValHisAlaLeuLys                              850855860                                                                     GATGAGCGGGCGGTGCACATCCTGGAGGACTTCGGGGCGCTGGCCGAT864                           AspGluArgAlaValHisIleLeuGluAspPheGlyAlaLeuAlaAsp                              865870875                                                                     CAGATCTCCGCCGAGGTGGGACGGCCGCTGACGCTCATCGCCGAGTCC912                           GlnIleSerAlaGluValGlyArgProLeuThrLeuIleAlaGluSer                              880885890                                                                     GACCTCAACAACCCGCGGCTGCTGTACCCGCGGGACGTCAACGGGTAC960                           AspLeuAsnAsnProArgLeuLeuTyrProArgAspValAsnGlyTyr                              895900905                                                                     GGGCTGGAAGGGCAGTGGAGCGACGACTTCCACCACGCCGTCCACGTC1008                          GlyLeuGluGlyGlnTrpSerAspAspPheHisHisAlaValHisVal                              910915920925                                                                  AACGTCACCGGCGAAACCACCGGCTACTACAGTGACTTCGACTCGCTG1056                          AsnValThrGlyGluThrThrGlyTyrTyrSerAspPheAspSerLeu                              930935940                                                                     GCCGCCCTCGCCAAGGTGCTCCGGGACGGCTTCTTCCACGACGGCAGC1104                          AlaAlaLeuAlaLysValLeuArgAspGlyPhePheHisAspGlySer                              945950955                                                                     TACTCCAGCTTCCGGGAACGCCACCACGGACGGCCGATTAATTTCAGC1152                          TyrSerSerPheArgGluArgHisHisGlyArgProIleAsnPheSer                              960965970                                                                     GCCGTACACCCAGCCGCCCTGGTGGTCTGTTCGCAGAACCACGACCAG1200                          AlaValHisProAlaAlaLeuValValCysSerGlnAsnHisAspGln                              975980985                                                                     ATCGGCAACCGTGCCACGGGGGACCGGCTCTCCCAGACCCTGCCGTAC1248                          IleGlyAsnArgAlaThrGlyAspArgLeuSerGlnThrLeuProTyr                              99099510001005                                                                GGAAGCCTGGCCCTCGCTGCGGTGCTGACCCTGACGGGACCCTTCACG1296                          GlySerLeuAlaLeuAlaAlaValLeuThrLeuThrGlyProPheThr                              101010151020                                                                  CCCATGCTGCTCATGGGCGAGGAGTACGGCGCCAGCACGCCGTGGCAG1344                          ProMetLeuLeuMetGlyGluGluTyrGlyAlaSerThrProTrpGln                              102510301035                                                                  TTTTTCACCTCGCACCCGGAGCCGGAGCTCGGCAAGGCCACCGCGGAG1392                          PhePheThrSerHisProGluProGluLeuGlyLysAlaThrAlaGlu                              104010451050                                                                  GGCCGGATCAAGGAGTTCGAGCGCATGGGGTGGGATCCCGCCGTCGTG1440                          GlyArgIleLysGluPheGluArgMetGlyTrpAspProAlaValVal                              105510601065                                                                  CCCGATCCCCAGGATCCTGAGACGTTCCGCCGGTCCAAGCTGGACTGG1488                          ProAspProGlnAspProGluThrPheArgArgSerLysLeuAspTrp                              1070107510801085                                                              GCGGAAGCCGCCGAAGGCGACCATGCCCGGCTGCTGGAGCTGTACCGT1536                          AlaGluAlaAlaGluGlyAspHisAlaArgLeuLeuGluLeuTyrArg                              109010951100                                                                  TCGCTCACCGCCCTGCGCCGCTCCACGCCGGACCTCACCAAGCTGGGC1584                          SerLeuThrAlaLeuArgArgSerThrProAspLeuThrLysLeuGly                              110511101115                                                                  TTCGAGGACACGCAGGTGGCGTTCGACGAGGACGCCCGCTGGCTGCGG1632                          PheGluAspThrGlnValAlaPheAspGluAspAlaArgTrpLeuArg                              112011251130                                                                  TTCCGCCGGGGTGGCGTGCAGGTGCTGCTCAACTTCTCGGAACAGCCC1680                          PheArgArgGlyGlyValGlnValLeuLeuAsnPheSerGluGlnPro                              113511401145                                                                  GTGAGCCTGGACGGGGCGGGCACGGCCCTGCTGCTGGCCACCGACGAC1728                          ValSerLeuAspGlyAlaGlyThrAlaLeuLeuLeuAlaThrAspAsp                              1150115511601165                                                              GCCGTCCGGCTAGAAGGTGAGCGTGCGGAACTCGGTCCGCTGAGCGCC1776                          AlaValArgLeuGluGlyGluArgAlaGluLeuGlyProLeuSerAla                              117011751180                                                                  GCCGTCGTCAGCGAC1791                                                           AlaValValSerAsp                                                               1185                                                                          (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 597 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       ThrHisThrTyrProArgGluAlaAlaLysProValLeuGlyProAla                              151015                                                                        ArgTyrAspValTrpAlaProAsnAlaGluSerValThrLeuLeuAla                              202530                                                                        GlyGlyGluArgTyrAlaMetGlnArgArgAlaGluThrGlyProGlu                              354045                                                                        AspAlaGlyTrpTrpThrAlaAlaGlyAlaProThrAspGlyAsnVal                              505560                                                                        AspTyrGlyTyrLeuLeuAspGlyAspGluThrProLeuProAspPro                              65707580                                                                      ArgThrArgArgGlnProAspGlyValHisAlaLeuSerArgThrPhe                              859095                                                                        AspProSerAlaTyrSerTrpGlnAspAspAlaTrpGlnGlyArgGlu                              100105110                                                                     LeuGlnGlyAlaValIleTyrGluLeuHisLeuGlyThrPheThrPro                              115120125                                                                     GluGlyThrLeuGluAlaAlaAlaGlyLysLeuAspTyrLeuAlaGly                              130135140                                                                     LeuGlyValAspPheIleGluLeuLeuProValAsnAlaPheAsnGly                              145150155160                                                                  ThrHisAsnTrpGlyTyrAspGlyValGlnTrpPheAlaValHisGlu                              165170175                                                                     AlaTyrGlyGlyProGluAlaTyrGlnArgPheValAspAlaAlaHis                              180185190                                                                     AlaAlaGlyLeuGlyValIleGlnAspValValTyrAsnHisLeuGly                              195200205                                                                     ProSerGlyAsnTyrLeuProArgPheGlyProTyrLeuLysGlnGly                              210215220                                                                     GluGlyAsnThrTrpGlyAspSerValAsnLeuAspGlyProGlySer                              225230235240                                                                  AspHisValArgArgTyrIleLeuAspAsnLeuAlaMetTrpLeuArg                              245250255                                                                     AspTyrArgValAspGlyLeuArgLeuAspAlaValHisAlaLeuLys                              260265270                                                                     AspGluArgAlaValHisIleLeuGluAspPheGlyAlaLeuAlaAsp                              275280285                                                                     GlnIleSerAlaGluValGlyArgProLeuThrLeuIleAlaGluSer                              290295300                                                                     AspLeuAsnAsnProArgLeuLeuTyrProArgAspValAsnGlyTyr                              305310315320                                                                  GlyLeuGluGlyGlnTrpSerAspAspPheHisHisAlaValHisVal                              325330335                                                                     AsnValThrGlyGluThrThrGlyTyrTyrSerAspPheAspSerLeu                              340345350                                                                     AlaAlaLeuAlaLysValLeuArgAspGlyPhePheHisAspGlySer                              355360365                                                                     TyrSerSerPheArgGluArgHisHisGlyArgProIleAsnPheSer                              370375380                                                                     AlaValHisProAlaAlaLeuValValCysSerGlnAsnHisAspGln                              385390395400                                                                  IleGlyAsnArgAlaThrGlyAspArgLeuSerGlnThrLeuProTyr                              405410415                                                                     GlySerLeuAlaLeuAlaAlaValLeuThrLeuThrGlyProPheThr                              420425430                                                                     ProMetLeuLeuMetGlyGluGluTyrGlyAlaSerThrProTrpGln                              435440445                                                                     PhePheThrSerHisProGluProGluLeuGlyLysAlaThrAlaGlu                              450455460                                                                     GlyArgIleLysGluPheGluArgMetGlyTrpAspProAlaValVal                              465470475480                                                                  ProAspProGlnAspProGluThrPheArgArgSerLysLeuAspTrp                              485490495                                                                     AlaGluAlaAlaGluGlyAspHisAlaArgLeuLeuGluLeuTyrArg                              500505510                                                                     SerLeuThrAlaLeuArgArgSerThrProAspLeuThrLysLeuGly                              515520525                                                                     PheGluAspThrGlnValAlaPheAspGluAspAlaArgTrpLeuArg                              530535540                                                                     PheArgArgGlyGlyValGlnValLeuLeuAsnPheSerGluGlnPro                              545550555560                                                                  ValSerLeuAspGlyAlaGlyThrAlaLeuLeuLeuAlaThrAspAsp                              565570575                                                                     AlaValArgLeuGluGlyGluArgAlaGluLeuGlyProLeuSerAla                              580585590                                                                     AlaValValSerAsp                                                               595                                                                           (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       AlaLysProValGlnGlyAlaGlyArgPheAspIleTrpAlaProGlu                              151015                                                                        AlaGlyThrVal                                                                  20                                                                            (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       ThrHisThrTyrProArgGluAlaAlaLysProValLeuGlyProAla                              151015                                                                        ArgTyrAspVal                                                                  20                                                                            (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       ProValGlnGlyAlaGlyArgPheAspIleTrpAlaProGluAlaGly                              151015                                                                        ThrValThrLeuLeu                                                               20                                                                            (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       LeuAspTrpAlaGluAlaSerAlaGlyAspHisAlaArgLeuLeuGlu                              151015                                                                        Leu                                                                           (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       GluPheGluArgMetGlyTrpAspProAlaValValProAspProGln                              151015                                                                        AspProGluThr                                                                  20                                                                            (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      ProValLeuGlyProAlaArgTyrAspValTrpAlaProAsnAlaGlu                              151015                                                                        SerValThrLeu                                                                  20                                                                            (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 2161 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 207..1994                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      GGCGCCGGGGGAGTGCTGGCGCTTGCCACCCGGCTCCCCTACGGGCTGGAACAGTCGGGC60                GGCTGGCGGGACACCGCCGTCGAGCTTGAAGCCGCCATGACGGACGAACTGACCGGCTCC120               ACTTTCGGGCCGGGACCGGCGGCGCTGTCAGAAGTCTTCCGGGCCTACCCGGTGGCCTTG180               TTGGTCCCCGCGACAGGAGGCAAGTCATGACGCAGCCCAACGATGCGGCCAAG233                      MetThrGlnProAsnAspAlaAlaLys                                                   600605                                                                        CCGGTGCAGGGAGCGGGGCGCTTCGATATCTGGGCGCCCGAGGCAGGC281                           ProValGlnGlyAlaGlyArgPheAspIleTrpAlaProGluAlaGly                              610615620                                                                     ACCGTAACGCTGCTGGCCGGCGGGGAGCGCTACGAGATGGGCCGCCGC329                           ThrValThrLeuLeuAlaGlyGlyGluArgTyrGluMetGlyArgArg                              625630635                                                                     CCCGGCAACGGGCCGGCGGACGAAGGCTGGTGGACGGCCGCGGATGCA377                           ProGlyAsnGlyProAlaAspGluGlyTrpTrpThrAlaAlaAspAla                              640645650                                                                     CCGACAGGCGCGGACGTGGACTACGGATACCTGCTCGACGGCGACGAA425                           ProThrGlyAlaAspValAspTyrGlyTyrLeuLeuAspGlyAspGlu                              655660665670                                                                  ATCCCGCTGCCGGACCCCCGGACCCGCCGCCAGCCCGAAGGCGTCCAT473                           IleProLeuProAspProArgThrArgArgGlnProGluGlyValHis                              675680685                                                                     GCCCTGTCCCGGACCTTCGACCCCGGCGCCCACCGCTGGCAGGACGCC521                           AlaLeuSerArgThrPheAspProGlyAlaHisArgTrpGlnAspAla                              690695700                                                                     GGGTGGCAGGGCAGGGAACTCCAGGGCTCCGTGATTTACGAACTCCAC569                           GlyTrpGlnGlyArgGluLeuGlnGlySerValIleTyrGluLeuHis                              705710715                                                                     ATCGGAACGTTCACGCCGGAAGGGACGCTGGACGCCGCCGCGGGCAAG617                           IleGlyThrPheThrProGluGlyThrLeuAspAlaAlaAlaGlyLys                              720725730                                                                     CTGGACTACCTCGCCGGCCTGGGCATCGACTTCATTGAGCTGCTGCCC665                           LeuAspTyrLeuAlaGlyLeuGlyIleAspPheIleGluLeuLeuPro                              735740745750                                                                  GTGAATGCCTTCAACGGCACGCACAACTGGGGCTACGACGGCGTCCAG713                           ValAsnAlaPheAsnGlyThrHisAsnTrpGlyTyrAspGlyValGln                              755760765                                                                     TGGTTTGCCGTGCATGAAGGCTACGGCGGGCCTGCGGCGTACCAGCGG761                           TrpPheAlaValHisGluGlyTyrGlyGlyProAlaAlaTyrGlnArg                              770775780                                                                     TTCGTGGATGCGGCCCACGCGGCCGGCCTCGGCGTCATCCAGGACGTG809                           PheValAspAlaAlaHisAlaAlaGlyLeuGlyValIleGlnAspVal                              785790795                                                                     GTCTACAACCACCTCGGGCCGAGCGGGAACTACCTCCCCAGGTACGGC857                           ValTyrAsnHisLeuGlyProSerGlyAsnTyrLeuProArgTyrGly                              800805810                                                                     CCGTACCTCAAGCACGGCGAAGGCAACACCTGGGGCGATTCGGTCAAC905                           ProTyrLeuLysHisGlyGluGlyAsnThrTrpGlyAspSerValAsn                              815820825830                                                                  CTGGACGGGCCGGGATCCGACCACGTCCGCCAGTACATCCTGGACAAC953                           LeuAspGlyProGlySerAspHisValArgGlnTyrIleLeuAspAsn                              835840845                                                                     GTGGCCATGTGGCTGCGCGACTACCGGGTGGACGGCCTCCGCCTGGAC1001                          ValAlaMetTrpLeuArgAspTyrArgValAspGlyLeuArgLeuAsp                              850855860                                                                     GCCGTCCACGCCCTGAAGGATGAGCGGGCCGTCCACATCCTGGAGGAG1049                          AlaValHisAlaLeuLysAspGluArgAlaValHisIleLeuGluGlu                              865870875                                                                     TTCGGCGCGCTGGCGGACGCCCTGTCGTCCGAAGGCGGCCGCCCGCTG1097                          PheGlyAlaLeuAlaAspAlaLeuSerSerGluGlyGlyArgProLeu                              880885890                                                                     ACCCTCATCGCCGAGTCCGACCTCAACAATCCGCGGCTGCTGTACCCC1145                          ThrLeuIleAlaGluSerAspLeuAsnAsnProArgLeuLeuTyrPro                              895900905910                                                                  CGGGATGTCAACGGCTACGGACTGGCCGGCCAGTGGAGCGACGACTTC1193                          ArgAspValAsnGlyTyrGlyLeuAlaGlyGlnTrpSerAspAspPhe                              915920925                                                                     CACCACGCCGTGCACGTCAACGTCAGCGGGGAAACCACCGGCTACTAC1241                          HisHisAlaValHisValAsnValSerGlyGluThrThrGlyTyrTyr                              930935940                                                                     AGCGACTTCGACTCGCTCGGAGCCCTCGCCAAGGTCCTGCGTGACGGG1289                          SerAspPheAspSerLeuGlyAlaLeuAlaLysValLeuArgAspGly                              945950955                                                                     TTCTTCCACGACGGCAGCTACTCCAGCTTCCGCGGCCGCTGCCACGGC1337                          PhePheHisAspGlySerTyrSerSerPheArgGlyArgCysHisGly                              960965970                                                                     CGGCCGATCAACTTCAGCGCCGTGCATCCGGCCGCGCTGGTGGTCTGC1385                          ArgProIleAsnPheSerAlaValHisProAlaAlaLeuValValCys                              975980985990                                                                  TCACAGAACCATGACCAGATCGGCAACCGGGCCACCGGGGACCGGCTG1433                          SerGlnAsnHisAspGlnIleGlyAsnArgAlaThrGlyAspArgLeu                              99510001005                                                                   TCCCAGTCACTTCCGTACGGCAGCCTGGCCCTGGCCGCCGTGCTGACC1481                          SerGlnSerLeuProTyrGlySerLeuAlaLeuAlaAlaValLeuThr                              101010151020                                                                  CTCACCGGTCCGTTCACGCCCATGCTGTTCATGGGAGAGGAATACGGG1529                          LeuThrGlyProPheThrProMetLeuPheMetGlyGluGluTyrGly                              102510301035                                                                  GCCACCACCCCGTGGCAGTTCTTCACCTCGCACCCTGAACCCGAGCTG1577                          AlaThrThrProTrpGlnPhePheThrSerHisProGluProGluLeu                              104010451050                                                                  GGCAAGGCCACGGCCGAGGGCAGGATCAGGGAGTTCGAGCGCATGGGG1625                          GlyLysAlaThrAlaGluGlyArgIleArgGluPheGluArgMetGly                              1055106010651070                                                              TGGGATCCCGCCGTCGTGCCCGATCCGCAGGATCCGGAGACCTTCACC1673                          TrpAspProAlaValValProAspProGlnAspProGluThrPheThr                              107510801085                                                                  CGCTCCAAACTGGACTGGGCGGAAGCGTCCGCCGGCGATCATGCCCGC1721                          ArgSerLysLeuAspTrpAlaGluAlaSerAlaGlyAspHisAlaArg                              109010951100                                                                  CTCCTGGAGCTGTACCGCTCGCTTATCACGCTGCGGCGGTCAACTCCG1769                          LeuLeuGluLeuTyrArgSerLeuIleThrLeuArgArgSerThrPro                              110511101115                                                                  GAGCTCGCGCGCCTGGGCTTTGCGGACACCGCCGTCGAGTTCGACGAC1817                          GluLeuAlaArgLeuGlyPheAlaAspThrAlaValGluPheAspAsp                              112011251130                                                                  GACGCCCGCTGGCTCCGTTATTGGCGCGGAGGCGTGCAGGTGGTGCTG1865                          AspAlaArgTrpLeuArgTyrTrpArgGlyGlyValGlnValValLeu                              1135114011451150                                                              AACTTCGCGGACCGTCCCATCAGCCTGGACCGGCCGGGAACCGCGCTG1913                          AsnPheAlaAspArgProIleSerLeuAspArgProGlyThrAlaLeu                              115511601165                                                                  CTGCTCGCCACCGACGACGCCGTCCGGATGGACGGAGTCCAGGTGGAG1961                          LeuLeuAlaThrAspAspAlaValArgMetAspGlyValGlnValGlu                              117011751180                                                                  CTGCCGCCGCTGAGCGCCGCGGTTCTGCGCGACTGAGCGTGCGCGCCTTCGGG2014                     LeuProProLeuSerAlaAlaValLeuArgAsp                                             11851190                                                                      GCGGGCGTCCTTCCGGTGACCGGATGCTGGACGCCCGCCCCGCAGCTCCACAGGCGCTGG2074              CAGGATGGAACGTATGACTTTTCTGGCAGCGGACAACCGCTACGAAACCATGCCATACCG2134              CCGCGTCGGACGCAGCGGGCTGAAGCT2161                                               (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 596 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      MetThrGlnProAsnAspAlaAlaLysProValGlnGlyAlaGlyArg                              151015                                                                        PheAspIleTrpAlaProGluAlaGlyThrValThrLeuLeuAlaGly                              202530                                                                        GlyGluArgTyrGluMetGlyArgArgProGlyAsnGlyProAlaAsp                              354045                                                                        GluGlyTrpTrpThrAlaAlaAspAlaProThrGlyAlaAspValAsp                              505560                                                                        TyrGlyTyrLeuLeuAspGlyAspGluIleProLeuProAspProArg                              65707580                                                                      ThrArgArgGlnProGluGlyValHisAlaLeuSerArgThrPheAsp                              859095                                                                        ProGlyAlaHisArgTrpGlnAspAlaGlyTrpGlnGlyArgGluLeu                              100105110                                                                     GlnGlySerValIleTyrGluLeuHisIleGlyThrPheThrProGlu                              115120125                                                                     GlyThrLeuAspAlaAlaAlaGlyLysLeuAspTyrLeuAlaGlyLeu                              130135140                                                                     GlyIleAspPheIleGluLeuLeuProValAsnAlaPheAsnGlyThr                              145150155160                                                                  HisAsnTrpGlyTyrAspGlyValGlnTrpPheAlaValHisGluGly                              165170175                                                                     TyrGlyGlyProAlaAlaTyrGlnArgPheValAspAlaAlaHisAla                              180185190                                                                     AlaGlyLeuGlyValIleGlnAspValValTyrAsnHisLeuGlyPro                              195200205                                                                     SerGlyAsnTyrLeuProArgTyrGlyProTyrLeuLysHisGlyGlu                              210215220                                                                     GlyAsnThrTrpGlyAspSerValAsnLeuAspGlyProGlySerAsp                              225230235240                                                                  HisValArgGlnTyrIleLeuAspAsnValAlaMetTrpLeuArgAsp                              245250255                                                                     TyrArgValAspGlyLeuArgLeuAspAlaValHisAlaLeuLysAsp                              260265270                                                                     GluArgAlaValHisIleLeuGluGluPheGlyAlaLeuAlaAspAla                              275280285                                                                     LeuSerSerGluGlyGlyArgProLeuThrLeuIleAlaGluSerAsp                              290295300                                                                     LeuAsnAsnProArgLeuLeuTyrProArgAspValAsnGlyTyrGly                              305310315320                                                                  LeuAlaGlyGlnTrpSerAspAspPheHisHisAlaValHisValAsn                              325330335                                                                     ValSerGlyGluThrThrGlyTyrTyrSerAspPheAspSerLeuGly                              340345350                                                                     AlaLeuAlaLysValLeuArgAspGlyPhePheHisAspGlySerTyr                              355360365                                                                     SerSerPheArgGlyArgCysHisGlyArgProIleAsnPheSerAla                              370375380                                                                     ValHisProAlaAlaLeuValValCysSerGlnAsnHisAspGlnIle                              385390395400                                                                  GlyAsnArgAlaThrGlyAspArgLeuSerGlnSerLeuProTyrGly                              405410415                                                                     SerLeuAlaLeuAlaAlaValLeuThrLeuThrGlyProPheThrPro                              420425430                                                                     MetLeuPheMetGlyGluGluTyrGlyAlaThrThrProTrpGlnPhe                              435440445                                                                     PheThrSerHisProGluProGluLeuGlyLysAlaThrAlaGluGly                              450455460                                                                     ArgIleArgGluPheGluArgMetGlyTrpAspProAlaValValPro                              465470475480                                                                  AspProGlnAspProGluThrPheThrArgSerLysLeuAspTrpAla                              485490495                                                                     GluAlaSerAlaGlyAspHisAlaArgLeuLeuGluLeuTyrArgSer                              500505510                                                                     LeuIleThrLeuArgArgSerThrProGluLeuAlaArgLeuGlyPhe                              515520525                                                                     AlaAspThrAlaValGluPheAspAspAspAlaArgTrpLeuArgTyr                              530535540                                                                     TrpArgGlyGlyValGlnValValLeuAsnPheAlaAspArgProIle                              545550555560                                                                  SerLeuAspArgProGlyThrAlaLeuLeuLeuAlaThrAspAspAla                              565570575                                                                     ValArgMetAspGlyValGlnValGluLeuProProLeuSerAlaAla                              580585590                                                                     ValLeuArgAsp                                                                  595                                                                           (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 2056 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 90..1883                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      GCCGGCTTCGGACCGGGGGCAGTGAAGATCGCCGACATCTTCCGGTCGTTCCCCGTTGCG60                CTGCTGGTGCCGCAGACAGGAGGAGAGTCATGACGCACACCTACCCGCGGGAA113                      MetThrHisThrTyrProArgGlu                                                      600                                                                           GCCGCGAAACCCGTCCTGGGCCCCGCACGCTACGACGTCTGGGCGCCC161                           AlaAlaLysProValLeuGlyProAlaArgTyrAspValTrpAlaPro                              605610615620                                                                  AACGCTGAATCCGTGACGCTGCTGGCCGGCGGGGAGCGCTACGCCATG209                           AsnAlaGluSerValThrLeuLeuAlaGlyGlyGluArgTyrAlaMet                              625630635                                                                     CAGCGCCGGGCCGAGACCGGGCCGGAGGACGCCGGCTGGTGGACCGCC257                           GlnArgArgAlaGluThrGlyProGluAspAlaGlyTrpTrpThrAla                              640645650                                                                     GCCGGCGCGCCTACGGATGGCAACGTGGACTACGGGTACCTTCTGGAC305                           AlaGlyAlaProThrAspGlyAsnValAspTyrGlyTyrLeuLeuAsp                              655660665                                                                     GGCGACGAAACACCGCTTCCGGATCCACGGACCCGCCGCCAGCCCGAC353                           GlyAspGluThrProLeuProAspProArgThrArgArgGlnProAsp                              670675680                                                                     GGCGTCCACGCCCTGTCCCGCACGTTCGACCCGTCCGCGTACAGCTGG401                           GlyValHisAlaLeuSerArgThrPheAspProSerAlaTyrSerTrp                              685690695700                                                                  CAGGACGACGCCTGGCAGGGCAGGGAACTGCAGGGCGCCGTCATCTAC449                           GlnAspAspAlaTrpGlnGlyArgGluLeuGlnGlyAlaValIleTyr                              705710715                                                                     GAGCTCCACCTCGGAACATTCACGCCCGAAGGGACGCTGGAGGCGGCC497                           GluLeuHisLeuGlyThrPheThrProGluGlyThrLeuGluAlaAla                              720725730                                                                     GCCGGAAAGCTGGACTACCTCGCCGGCTTGGGCGTCGACTTCATCGAG545                           AlaGlyLysLeuAspTyrLeuAlaGlyLeuGlyValAspPheIleGlu                              735740745                                                                     CTGCTGCCGGTGAACGCTTTCAACGGCACGCACAACTGGGGTTACGAC593                           LeuLeuProValAsnAlaPheAsnGlyThrHisAsnTrpGlyTyrAsp                              750755760                                                                     GGTGTCCAGTGGTTCGCTGTGCACGAGGCATACGGCGGGCCGGAAGCG641                           GlyValGlnTrpPheAlaValHisGluAlaTyrGlyGlyProGluAla                              765770775780                                                                  TACCAGCGGTTCGTCGACGCCGCCCACGCCGCAGGCCTTGGCGTGATC689                           TyrGlnArgPheValAspAlaAlaHisAlaAlaGlyLeuGlyValIle                              785790795                                                                     CAGGACGTGGTCTACAACCACCTCGGCCCCAGCGGGAACTACCTGCCG737                           GlnAspValValTyrAsnHisLeuGlyProSerGlyAsnTyrLeuPro                              800805810                                                                     CGGTTCGGGCCGTACCTCAAGCAGGGCGAGGGTAACACGTGGGGCGAC785                           ArgPheGlyProTyrLeuLysGlnGlyGluGlyAsnThrTrpGlyAsp                              815820825                                                                     TCGGTGAACCTGGACGGGCCCGGCTCCGACCATGTGCGCCGGTACATC833                           SerValAsnLeuAspGlyProGlySerAspHisValArgArgTyrIle                              830835840                                                                     CTGGACAACCTGGCCATGTGGCTGCGTGACTACCGGGTGGACGGCCTG881                           LeuAspAsnLeuAlaMetTrpLeuArgAspTyrArgValAspGlyLeu                              845850855860                                                                  CGGCTGGACGCCGTCCACGCCCTGAAGGATGAGCGGGCGGTGCACATC929                           ArgLeuAspAlaValHisAlaLeuLysAspGluArgAlaValHisIle                              865870875                                                                     CTGGAGGACTTCGGGGCGCTGGCCGATCAGATCTCCGCCGAGGTGGGA977                           LeuGluAspPheGlyAlaLeuAlaAspGlnIleSerAlaGluValGly                              880885890                                                                     CGGCCGCTGACGCTCATCGCCGAGTCCGACCTCAACAACCCGCGGCTG1025                          ArgProLeuThrLeuIleAlaGluSerAspLeuAsnAsnProArgLeu                              895900905                                                                     CTGTACCCGCGGGACGTCAACGGGTACGGGCTGGAAGGGCAGTGGAGC1073                          LeuTyrProArgAspValAsnGlyTyrGlyLeuGluGlyGlnTrpSer                              910915920                                                                     GACGACTTCCACCACGCCGTCCACGTCAACGTCACCGGCGAAACCACC1121                          AspAspPheHisHisAlaValHisValAsnValThrGlyGluThrThr                              925930935940                                                                  GGCTACTACAGTGACTTCGACTCGCTGGCCGCCCTCGCCAAGGTGCTC1169                          GlyTyrTyrSerAspPheAspSerLeuAlaAlaLeuAlaLysValLeu                              945950955                                                                     CGGGACGGCTTCTTCCACGACGGCAGCTACTCCAGCTTCCGGGAACGC1217                          ArgAspGlyPhePheHisAspGlySerTyrSerSerPheArgGluArg                              960965970                                                                     CACCACGGACGGCCGATTAATTTCAGCGCCGTACACCCAGCCGCCCTG1265                          HisHisGlyArgProIleAsnPheSerAlaValHisProAlaAlaLeu                              975980985                                                                     GTGGTCTGTTCGCAGAACCACGACCAGATCGGCAACCGTGCCACGGGG1313                          ValValCysSerGlnAsnHisAspGlnIleGlyAsnArgAlaThrGly                              9909951000                                                                    GACCGGCTCTCCCAGACCCTGCCGTACGGAAGCCTGGCCCTCGCTGCG1361                          AspArgLeuSerGlnThrLeuProTyrGlySerLeuAlaLeuAlaAla                              1005101010151020                                                              GTGCTGACCCTGACGGGACCCTTCACGCCCATGCTGCTCATGGGCGAG1409                          ValLeuThrLeuThrGlyProPheThrProMetLeuLeuMetGlyGlu                              102510301035                                                                  GAGTACGGCGCCAGCACGCCGTGGCAGTTTTTCACCTCGCACCCGGAG1457                          GluTyrGlyAlaSerThrProTrpGlnPhePheThrSerHisProGlu                              104010451050                                                                  CCGGAGCTCGGCAAGGCCACCGCGGAGGGCCGGATCAAGGAGTTCGAG1505                          ProGluLeuGlyLysAlaThrAlaGluGlyArgIleLysGluPheGlu                              105510601065                                                                  CGCATGGGGTGGGATCCCGCCGTCGTGCCCGATCCCCAGGATCCTGAG1553                          ArgMetGlyTrpAspProAlaValValProAspProGlnAspProGlu                              107010751080                                                                  ACGTTCCGCCGGTCCAAGCTGGACTGGGCGGAAGCCGCCGAAGGCGAC1601                          ThrPheArgArgSerLysLeuAspTrpAlaGluAlaAlaGluGlyAsp                              1085109010951100                                                              CATGCCCGGCTGCTGGAGCTGTACCGTTCGCTCACCGCCCTGCGCCGC1649                          HisAlaArgLeuLeuGluLeuTyrArgSerLeuThrAlaLeuArgArg                              110511101115                                                                  TCCACGCCGGACCTCACCAAGCTGGGCTTCGAGGACACGCAGGTGGCG1697                          SerThrProAspLeuThrLysLeuGlyPheGluAspThrGlnValAla                              112011251130                                                                  TTCGACGAGGACGCCCGCTGGCTGCGGTTCCGCCGGGGTGGCGTGCAG1745                          PheAspGluAspAlaArgTrpLeuArgPheArgArgGlyGlyValGln                              113511401145                                                                  GTGCTGCTCAACTTCTCGGAACAGCCCGTGAGCCTGGACGGGGCGGGC1793                          ValLeuLeuAsnPheSerGluGlnProValSerLeuAspGlyAlaGly                              115011551160                                                                  ACGGCCCTGCTGCTGGCCACCGACGACGCCGTCCGGCTAGAAGGTGAG1841                          ThrAlaLeuLeuLeuAlaThrAspAspAlaValArgLeuGluGlyGlu                              1165117011751180                                                              CGTGCGGAACTCGGTCCGCTGAGCGCCGCCGTCGTCAGCGAC1883                                ArgAlaGluLeuGlyProLeuSerAlaAlaValValSerAsp                                    11851190                                                                      TGACGTTTTCTTGGGGGCGGCGTCCACCGCCGGTGACCGGATGGTGGACGTCCGCCCCGA1943              AGCCTCGGCGCGGCTGGCAGGATGGAACGCATGACTTATGTGGCCTCGGACACCCGCTAC2003              GACACCATGCCCTACCGCCGCGTCGGACGCAGCGGCCTCAAACTGCCGGCCAT2056                     (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 598 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      MetThrHisThrTyrProArgGluAlaAlaLysProValLeuGlyPro                              151015                                                                        AlaArgTyrAspValTrpAlaProAsnAlaGluSerValThrLeuLeu                              202530                                                                        AlaGlyGlyGluArgTyrAlaMetGlnArgArgAlaGluThrGlyPro                              354045                                                                        GluAspAlaGlyTrpTrpThrAlaAlaGlyAlaProThrAspGlyAsn                              505560                                                                        ValAspTyrGlyTyrLeuLeuAspGlyAspGluThrProLeuProAsp                              65707580                                                                      ProArgThrArgArgGlnProAspGlyValHisAlaLeuSerArgThr                              859095                                                                        PheAspProSerAlaTyrSerTrpGlnAspAspAlaTrpGlnGlyArg                              100105110                                                                     GluLeuGlnGlyAlaValIleTyrGluLeuHisLeuGlyThrPheThr                              115120125                                                                     ProGluGlyThrLeuGluAlaAlaAlaGlyLysLeuAspTyrLeuAla                              130135140                                                                     GlyLeuGlyValAspPheIleGluLeuLeuProValAsnAlaPheAsn                              145150155160                                                                  GlyThrHisAsnTrpGlyTyrAspGlyValGlnTrpPheAlaValHis                              165170175                                                                     GluAlaTyrGlyGlyProGluAlaTyrGlnArgPheValAspAlaAla                              180185190                                                                     HisAlaAlaGlyLeuGlyValIleGlnAspValValTyrAsnHisLeu                              195200205                                                                     GlyProSerGlyAsnTyrLeuProArgPheGlyProTyrLeuLysGln                              210215220                                                                     GlyGluGlyAsnThrTrpGlyAspSerValAsnLeuAspGlyProGly                              225230235240                                                                  SerAspHisValArgArgTyrIleLeuAspAsnLeuAlaMetTrpLeu                              245250255                                                                     ArgAspTyrArgValAspGlyLeuArgLeuAspAlaValHisAlaLeu                              260265270                                                                     LysAspGluArgAlaValHisIleLeuGluAspPheGlyAlaLeuAla                              275280285                                                                     AspGlnIleSerAlaGluValGlyArgProLeuThrLeuIleAlaGlu                              290295300                                                                     SerAspLeuAsnAsnProArgLeuLeuTyrProArgAspValAsnGly                              305310315320                                                                  TyrGlyLeuGluGlyGlnTrpSerAspAspPheHisHisAlaValHis                              325330335                                                                     ValAsnValThrGlyGluThrThrGlyTyrTyrSerAspPheAspSer                              340345350                                                                     LeuAlaAlaLeuAlaLysValLeuArgAspGlyPhePheHisAspGly                              355360365                                                                     SerTyrSerSerPheArgGluArgHisHisGlyArgProIleAsnPhe                              370375380                                                                     SerAlaValHisProAlaAlaLeuValValCysSerGlnAsnHisAsp                              385390395400                                                                  GlnIleGlyAsnArgAlaThrGlyAspArgLeuSerGlnThrLeuPro                              405410415                                                                     TyrGlySerLeuAlaLeuAlaAlaValLeuThrLeuThrGlyProPhe                              420425430                                                                     ThrProMetLeuLeuMetGlyGluGluTyrGlyAlaSerThrProTrp                              435440445                                                                     GlnPhePheThrSerHisProGluProGluLeuGlyLysAlaThrAla                              450455460                                                                     GluGlyArgIleLysGluPheGluArgMetGlyTrpAspProAlaVal                              465470475480                                                                  ValProAspProGlnAspProGluThrPheArgArgSerLysLeuAsp                              485490495                                                                     TrpAlaGluAlaAlaGluGlyAspHisAlaArgLeuLeuGluLeuTyr                              500505510                                                                     ArgSerLeuThrAlaLeuArgArgSerThrProAspLeuThrLysLeu                              515520525                                                                     GlyPheGluAspThrGlnValAlaPheAspGluAspAlaArgTrpLeu                              530535540                                                                     ArgPheArgArgGlyGlyValGlnValLeuLeuAsnPheSerGluGln                              545550555560                                                                  ProValSerLeuAspGlyAlaGlyThrAlaLeuLeuLeuAlaThrAsp                              565570575                                                                     AspAlaValArgLeuGluGlyGluArgAlaGluLeuGlyProLeuSer                              580585590                                                                     AlaAlaValValSerAsp                                                            595                                                                           (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      TTYGAYATHTGGGCNCC17                                                           (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      GTAAAACGACGGCCAGT17                                                           (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      ATGGGNTGGGAYCCNGC17                                                           (2) INFORMATION FOR SEQ ID NO:18:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                      TAYGAYGTNTGGGC14                                                              __________________________________________________________________________

We claim:
 1. A recombinant enzyme which releases trehalose from anon-reducing saccharide having a trehalose structure as an end unit andhaving a degree of glucose polymerization of 3 or higher, saidrecombinant enzyme being obtained from a recombinant host microorganismby expressing said recombinant enzyme from a DNA sequence which encodessaid recombinant enzyme and which is derived from a microorganismselected from the genera consisting of Rhizobium, Arthrobacter,Brevibacterium, and Micrococcus, and wherein said recombinant enzyme hasan amino acid sequence selected from the group consisting of SEQ IDNO:2, SEQ ID NO:4, and a variant thereof where one or more amino acidresidues in SEQ ID NO:2 or SEQ ID NO:4 are deleted, replaced with adifferent amino acid, or supplemented with one or more amino acids,which variant is a functional equivalent of the recombinant enzyme ofSEO ID NO:2 or SEO ID NO:4 and has substantially the same activity. 2.The recombinant enzyme as claimed in claim 1, which has the followingphysicochemical properties of:(1) Molecular weight About 57,000-68,000daltons on sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE); and (2) Isoelectric point (pI) About 3.3-4.6 onisoelectrophoresis.
 3. The recombinant enzyme as claimed in claim 1,which is encoded by a DNA having a base sequence selected from the groupconsisting of SEQ ID NOs:1 and 3 that initiate from the 5'-terminus, andvariants thereof where one or more bases in SEO ID NO:1 or 3 arereplaced with different bases by means of the degeneracy of the geneticcode without altering the corresponding amino acid sequences of SEQ IDNO:2 and SEQ ID NO:4, and complementary base sequences thereof.
 4. Therecombinant enzyme as claimed in claim 3, wherein said DNA has a basesequence of a variant where one or more bases in SEQ ID NOs:1 and 3 arereplaced with different bases by means of the degeneracy of the geneticcode without altering the corresponding amino acid sequences of SEQ IDNOs:2 and
 4. 5. The recombinant enzyme as claimed in claim 1, which isencoded by a DNA having a base sequence selected from the groupconsisting of SEQ ID NOs:11 and
 13. 6. The recombinant enzyme accordingto claim 1, wherein said recombinant enzyme has the amino acid sequenceof a variant where one amino acid residue in SEQ ID NO:2 or SEQ ID NO:4is deleted, replaced with a different amino acid or supplemented with asingle amino acid without substantially altering the activity of saidrecombinant enzyme.
 7. A recombinant enzyme which releases trehalosefrom a non-reducing saccharide having a trehalose structure as an endunit and having a degree of glucose polymerization of 3 or higher, saidrecombinant enzyme being obtained from a recombinant host microorganismby expressing said recombinant enzyme from a DNA sequence which encodessaid recombinant enzyme and which is derived from a microorganismselected from the genera consisting of Rhizobium, Arthrobacter,Brevibacterium, and Micrococcus, wherein said recombinant enzyme has anamino acid sequence selected from SEQ ID NO:4 and a variant thereofwhere one amino acid residue in SEQ ID NO:4 is deleted, replaced with adifferent amino acid or supplemented with a single amino acid withoutsubstantially altering the activity of said recombinant enzyme.
 8. Therecombinant enzyme according to claim 7, wherein said recombinant enzymehas the amino acid sequence of SEQ ID NO:4.
 9. The recombinant enzymeaccording to claim 7, wherein said recombinant enzyme has the amino acidsequence of a variant where one amino acid residue in SEQ ID NO:4 isdeleted, replaced with a different amino acid or supplemented with asingle amino acid without substantially altering the activity of saidrecombinant enzyme.
 10. The recombinant enzyme according to claim 7,which has the following physicochemical properties of:(1) Molecularweight About 57,000-68,000 daltons on sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE); and (2) Isoelectric point(pI) About 3.3-4.6 on isoelectrophoresis.
 11. The recombinant enzymeaccording to claim 7, which is encoded by a DNA sequence selected fromthe group consisting of SEQ ID NO:3, a variant thereof where one or morebases are replaced with different bases by means of the degeneracy ofthe genetic code without altering the corresponding amino acid sequenceof SEQ ID NO:4, and a complementary base sequence thereof.
 12. Therecombinant enzyme according to claim 11, wherein said recombinantenzyme is encoded by a DNA sequence having the sequence of SEQ ID NO:3.13. The recombinant enzyme according to claim 11, wherein saidrecombinant enzyme is encoded by a DNA sequence having the sequence of avariant where one or more bases in SEQ ID NO:3 are replaced withdifferent bases by means of the degeneracy of the genetic code withoutaltering the corresponding amino acid of SEQ ID NO:4.
 14. A recombinantenzyme which releases trehalose from a non-reducing saccharide having atrehalose structure as an end unit and having a degree of glucosepolymerization of 3 or higher, said recombinant enzyme being obtainedfrom a recombinant host microorganism by expressing said recombinantenzyme from a DNA sequence which encodes said recombinant enzyme andwhich is derived from a microorganism selected from the generaconsisting of Rhizobium, Arthrobacter, Brevibacterium, and Micrococcus,wherein said recombinant enzyme has an amino acid sequence selected fromSEQ ID NO:2 and a variant thereof where one amino acid residue in SEQ IDNO:2 is deleted, replaced with a different amino acid or supplementedwith a single amino acid without substantially altering the activity ofsaid recombinant enzyme.
 15. The recombinant enzyme according to claim14, wherein said recombinant enzyme has the amino acid sequence of SEQID NO:2.
 16. The recombinant enzyme according to claim 14, wherein saidrecombinant enzyme has the amino acid sequence of a variant where oneamino acid residue in SEQ ID NO:2 is deleted, replaced with a differentamino acid or supplemented with a single amino acid withoutsubstantially altering the activity of said recombinant enzyme.
 17. Therecombinant enzyme according to claim 14, which has the followingphysicochemical properties of:(1) Molecular weight About 57,000-68,000daltons on sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE); and (2) Isoelectric point (pI) About 3.3-4.6 onisoelectrophoresis.
 18. The recombinant enzyme according to claim 14,which is encoded by a DNA sequence selected from the group consisting ofSEQ ID NO:1, a variant thereof where one or more bases are replaced withdifferent bases by means of the degeneracy of the genetic code withoutaltering the corresponding amino acid sequence of SEQ ID NO:2, and acomplementary base sequence thereof.
 19. The recombinant enzymeaccording to claim 18, wherein said recombinant enzyme is encoded by aDNA sequence having the sequence of SEQ ID NO:1.
 20. The recombinantenzyme according to claim 18, wherein said recombinant enzyme is encodedby a DNA sequence having the sequence of a variant where one or morebases in SEQ ID NO:1 are replaced with different bases by means of thedegeneracy of the genetic code without altering the corresponding aminoacid of SEQ ID NO:2.